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”HE-"SW

                                                                                                                             

3 1293 009142

This is to certify that the

dissertation entitled

Supplemental Microbial Phytase Improves Utilization
of Phytate Phosphorus and Other Minerals by Young Pigs

presented by

Xingen Lei

has been accepted towards fulfillment
of the requirements for

Ph. D. Animal Science

degree in

 

 

flow/{O/flflw/

K/M '1 jOl‘ professor

 

 

Date/VOV. 24) /?Z/?/

MS U is an Affirmative Action/Equal Opportunity Instimlinn 0— 12771

LIBRARY

Michigan State
University

 

PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date due.

DATE DUE DATE DUE DATE DUE

 

 

. r, 7 ,7— 4
EDVQE134‘

 

 

tier 2 31995

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

MSU Is An Affirmative Action/Equal Opportunity Institution
c:\clrc\datodm.pm3—o. 1

SUPPLEMENTAL MICROBIAL PHYTASE IMPROVES UTILIZATION OF
PHYTATE PHOSPHORUS AND OTHER MINERALS BY YOUNG PIGS

BY

Xingen Lei

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Animal Science

1992

ABSTRACT

SUPPLEMENTAL MICROBIAL PHYTASE IMPROVES UTILIZATION
OF PHYTATE PHOSPHORUS AND OTHER MINERALS BY YOUNG PIGS

BY

Xingen Lei

The low bioavailability of phytate-P in plant feeds to
pigs contributes to diet expense of supplemental P and to P
pollution in areas of intensive animal agriculture. The
objectives of this research were to determine effects of
supplemental Asperqillus niqgr phytase on utilization of
phytate-P, Zn, and Ca, and interactions of phytase, Ca, Zn,
and vitamin D in swine diets. Four major consecutive
experiments were conducted with 258 weanling pigs. Variable
levels of phytase, Zn, Ca, and vitamin D were incorporated
into corn-soybean meal basal diets (BD) without added
inorganic forms of P and(or) Zn in seven different trials.
Response criteria including growth performance, plasma
mineral concentrations and alkaline phosphatase (AP)
activity, and mineral balance were repeatedly measured over
time. Linear increases in weight gain, feed intake, and
plasma inorganic P (P) concentrations were observed with

increased phytase activity from O to 750 phytase units

Xingen Lei
(PU)/g BD. Responses of weight gain, gain/feed, and plasma
AP activity were maximized at 1,200 PU/g BD. Concentrations
of plasma P continued to increase and plasma Ca decreased
linearly with increases in enzyme activity from 750 to 1,350
PU/g BD. Supplementing the BD with 750 PU/g increased P
retention by 50% and reduced fecal P excretion by 42%, and
1200 or 1350 PU/g BD improved these two measures further.
One thousand PU were equal in effect to .91 mg P from
calcium phosphate. Pigs receiving 1,350 PU/g BD maintained
normal concentrations of plasma Zn and P and normal rates
and efficiency of gain. Supplemental phytase improved
dietary Ca utilization, but did not affect plasma
concentrations of Mg, Cu, or Fe. Only a reduced dietary Ca
(.5%) level allowed supplemental phytase to produce all the
above improvements. Normal to high dietary Ca (.9%) markedly
decreased the efficacy of phytase, which was partially
alleviated by raising dietary vitamin D. In conclusion,
supplementing corn-soybean meal diets of weanling pigs with
A. niger phytase at 1,200 PU/g appeared to maximize phytate-
P utilization at a reduced diet Ca level, and essentially

obviated the need for inorganic P addition.

ACKNOWLEDGEMENTS

In completing my Ph.D., I am deeply grateful to my
major professor, Dr. E. R. Miller. He has always given
freely of his time and knowledge to teach me not only swine
nutrition but also the truth of life. He has educated me not
only in his office but also at spots of research. He has
shown a great deal of care not only for my current progress
but also for my future career. The same appreciation goes to
my former adviser, Professor F. Yang of Sichuan Agricultural
University, who has also served on my current committee. His
global views on animal nutrition, extreme openness, and
great expectations have inspired me to pursue advanced
research. I am very proud of working under two outstanding
scientists during the last decade.

Dr. M. T. Yokoyama has served as a co-advisor,
encouraged me to screen microbes of high phytase producers,
and supported all my activities during the course of my
study. He has been very patient, open-minded, and generous
to me. Dr. D. E. Ullrey has spent a lot of time to guide me
in designing research and concisely interpreting results. I

have been very impressed by his dedication, spirit, and

IV

wisdom. Dr. M. G. Hogberg has offered me generous support
for all my activities plus great smiling encouragement.

Dr. W. G. Pond of USDA, Children's Nutrition Research Center
at Houston, Texas has offered me very challenging
comprehensive questions and critical views on the journal
manuscripts of my research. I am deeply grateful to all of
them and very proud of having such excellent scholars on my
committee.

A special appreciation goes to Dr. P. K. Ku. He has
been involved in all my research projects, shared his
knowledge and experience with me, and helped me in analyzing
samples. Appreciation is also extended to Drs. I. V. Mao, T.
Chang, and J. Gill of the Animal Science Department, and
Drs. D. R. Romsos and M. H. Zile of the Food Science and
Human Nutrition Department for their time and knowledge
contributed to my program. Dr. S. Hengemuehle deserves a
special mention for help in the microbiological laboratory.
I also appreciate the interactions and friendships that I
have shared with my fellow graduate students. I thank the
Swine Teaching and Research Center staff, and the
Departmental secretaries and technicians for their generous
support.

I am grateful to Dr. D. Isleib, director of the
Institute of International Agriculture. He and his staff
have willingly offered me chances to participate in many of

the exchange programs between Sichuan Agricultural

V

University and Michigan State University. Thanks go far back
home to my former Professors D. Duanmu, K. R. Chen, J. R.
Wang, S. R. Zhang, and W. R. Tang for their continuous
support of my study.

Finally, I am indebted to my parents and other family
members for their love, support, and sacrifice during our
ten years apart. My wife, Li Li has continuously provided me
with encouragement, support and understanding at the time
she is also building her own career. I treasure the life and

the joy we have been sharing from our son, Rayleigh.

VI

Page

TABLE OF CONTENTS

LIST OF TABLES. . . . . . . . . . . . . . .

LIST OF ABBREVIATIONS. . . . . . . . .

INTRODUCTION. . . . . . . . . . . .

LITERATURE REVIEW . . . . . . . . .

Phosphorus . . . .

Phosphorus has versatile functions in animal body.

Phosphorus metabolism depends on Ca, vitamin D, and

other factors . . . .
Phosphorus supplements in diets are very costly

Phytate .
Phytate stores phosphate and energy in plant seeds .

Phytate represents most of phosphorus in cereals and

legumes .
Phytate is fairly indigestible by simple-stomached
animals. . . . . .
Phytate utilization varies with dietary factors
and age . . . . .

Phytate is an antinutritional factor . .
Phytate content in foods can be reduced
by processing . . . . .

Phytase . . . .

Phytase initiates phytate degradation . . .
Phytase activity occurs in microorganisms, plants,
and animal tissues . .
Phytase releases phytate-phosphorus. from. plant
foods in vitro . . .
Phytase supplementation in diets improves phytate-
phosphorus bioavailability to poultry . .
Phytase supplementation in diets improves phytate-
phosphorus bioavailability to swine . .
Phytase supplementation in diets improves phytate
degradation in humans . . . . . .

Summary . . . .

VII

XI

XIV

9

.10

12
I4

16

18
18

20

22

24

26

29

30

EXPERIMENTAL SERIES I LINEAR IMPROVEMENTS IN PHYTATE
PHOSPHORUS BIOAVAILABILITY BY
SUPPLEMENTAL DIETARY PHYTASE .
ABSTRACT . . . . . . .
Introduction . . . . . . .

Materials and Methods . . . . .

Phytase . . . . . . .
Animals and Treatments . . . . . .
Basal Diets . . . . . . . .
Sample Collection and Measurements . . .
Assays . . . . . . . . .
Statistics . . . . . . . .
Results . . . . . . . . .
Experiment 1.1 . . . . . . .
Balance of P and Ca. . . . .
Serum Inorganic P and Ca Concentrations, and
Alkaline Phosphatase Activity . . . .
Weight Gain . . . . . . .
Experiment 1.2 . . . . . .

Plasma Inorganic P and Ca Concentrations, and
Alkaline Phosphatase Activity . . .
Weight Gain, Feed Intake, and Gain/Feed . .
Discussion . . . . . . . .

Implications . . . . . .

EXPERIMENTAL SERIES II MAXIMAL IMPROVEMENTS IN PHYTATE

PHOSPHORUS BIOAVAILABILITY BY
SUPPLEMENTAL DIETARY PHYTASE

ABSTRACT . . . . . .
Introduction . . . . . . .

Materials and Methods . . . . .
Phytase and Diets . . . . . .
Animals and Treatments . . . . .
Sample Collection and Measurements . .
Assays . . . . . . . . .
Statistics . . . . . . . .

Results . . . . . . . .
Experiment 2.1 .
Daily Gain, Daily Feed Intake and Gain/Feed .
Plasma Inorganic P and Ca Concentrations and
Alkaline Phosphatase Activity . . . .
Plasma Mg, Cu, Fe, and Zn Concentrations . .

VIII

32

32

33

35
35
35
36
39
39
39

4O
40
4O

43
45
46

46
48

50

57

58

58

6O

61
61
61
64
64
65

66
66
66

68
7O

Breakpoints of Dietary Phytase Activity . . . 70

Experiment 2.2 . . . . . . . 72
Balance of P and Ca . . . . . . 72
Plasma Inorganic P, Ca, and Zn Concentrations, and

Alkaline Phosphatase Activity . . . . 77
Relationship between Urinary P and Ca Excretion
and Plasma Inorganic P and Ca Concentration . . 77
Daily Gain . . . . . . . . 79
Discussion . . . . . . . . 79
Implications . . . . . . . 85

EXPERIMENTAL SERIES III SIMULTANEOUS IMPROVEMENTS IN
PHYTATE PHOSPHORUS AND ZINC
BIOAVAILABILITY BY SUPPLEMENTAL

DIETARY PHYTASE . . . . 86
ABSTRACT . . . . . . . . 86
Introduction . . . . . . . 87
Materials and Methods . . . . . 89
Phytase, Zinc, Diets. . . . . 89
Animals and Treatments . . . . . 91
Sample Collection and Measurements . . . 91
Assays . . . . . . . 92
Statistics . . . . . . . 92
Results . . . . . . . . . . 93
Experiment 3.1 . . . . . . . 93
Main Effects . . . . . . . . 93
Growth Performance . . . . 95
Plasma Alkaline Phosphatase Activity . . 97
Plasma Concentrations of Minerals . . . 97
Experiment 3. 2 . . . . . . 101
Mineral Balance . . . 101
Plasma Concentrations of Minerals and Alkaline
Phosphatase Activity . . . . . 103
Weight Gain . . . . . . . 106
Discussion . . . . . . . 106
Implications . . . . . . . 114

EXPERIMENTAL SERIES IV INTERACTIONS OF PHYTASE, VITAMIN D,
AND CALCIUM ON PHYTATE PHOSPHORUS
UTILIZATION . . . . . . 115
ABSTRACT . . . . . . . . 115

IX

Introduction

Materials and Methods

Experimental Design .

Phytase and Diets

Animals

Sample Collection and Mea
Statistics

Results

Main Effects and Interaction of Treatments
Daily Gain, Daily Feed Intake and Gain/Feed
Plasma Inorganic P and Ca Concentrations and

surements

Alkaline Phosphatase Activity .

Correlations among Various Response Measures
Discussion
Implications

GENERAL DISCUSSION
Progress in this Research .
Application of this Research

Limitation of this Research

BIBLIOGRAPHY

116
118
118
118
118
119
119
122
122
124

127
129

130

135

136

136

139

140

142

 

 

Table

Table

Table

Table

Table

Table

Table

Table

Table

Table

1.7.

2.2.

2.3.

LIST OF TABLES

Page
Composition and nutritive values of
basal diets . . . . . . . . . . . . 37
Analyzed dietary Ca and P concentrations of
experimental diets . . . . . . . . . .38

Balance of P and Ca in pigs fed diet with or
without supplemental microbial phytase . . . 42

Serum inorganic P and Ca concentrations, and
serum alkaline phosphatase activity of pigs

fed diet with or without supplemental

microbial phytase in experiment 1.1 . . . . .44

Daily gains of pigs fed diet with or without
supplemental microbial phytase in
experiment 1.1 . . . . . . . . . . .45

Plasma inorganic P and Ca concentrations, and
plasma alkaline phosphatase activity of

pigs receiving graded dietary levels of
supplemental microbial phytase activity in
experiment 1.2 . . . . . . . . . . . 47

Daily gain, feed intake, and gain/feed of pigs
receiving graded dietary levels of

supplemental microbial phytase activity in
experiment 1.2 . . . . . . . . . . .49

Composition and nutritive values of the basal
diet and basal diet supplemented with mono-
dibasic calcium phosphate (MDCaP) . . . 62

Analyzed dietary concentrations of P, Ca, and
other elements . . . . . . . . . 63

Daily gain, feed intake, and feed efficiency of

pigs receiving graded levels of supplemental
microbial phytase activity or supplemental
mono-dibasic calcium phosphate (MDCaP) in

the diet in experiment 2.1 . . . . . . . 67

XI

Table

Table

Table

Table

Table

Table

Table

Table

Table

Table

Plasma inorganic P and Ca concentrations, and
alkaline phosphatase activity of pigs

receiving graded levels of supplemental
microbial phytase activity or supplemental
mono-dibasic calcium phosphate (MDCaP) in

the diet in experiment 2.1 . . . . . . 69

Plasma concentrations of Mg, Cu, Fe, and Zn in
pigs receiving graded levels of supplemental
microbial phytase activity or supplemental
mono-dibasic calcium phosphate (MDCaP) in
the diet in experiment 2.1 . . . . . . 71

Regression coefficients of different measures
with dietary phytase activity
in experiment 2.1 . . . . . . . . 73

Breakpoints of dietary phytase activity for
different response measures and the

comparison of these maximum responses with
those of the control pigs receiving
supplemental mono-dibasic calcium phosphate

in the diet in experiment 2.1 . . . . . 74

Balance of P and Ca in pigs fed the basal diet
supplemented with or without the optimal dose
of microbial phytase or mono-dibasic calcium
phosphate (MDCaP) in experiment 2.2 . . . .75

Plasma inorganic P, Ca, and Zn concentrations
and alkaline phosphatase activity of pigs fed
the basal diet supplemented with or without
the optimal dose of microbial phytase or
mono-dibasic calcium phosphate (MDCaP) in
experiment 2.2 . . . . . . . . 78

Composition and nutrient values of
the basal diet . . . . . . . . . .90

Significances and standard errors of mean
differences of main effects of dietary

phytase and zinc, and their interactions on
various measures . . . . . . . .94

Daily feed intake, weight gain, and gain/feed
of pigs receiving different dietary levels of
supplemental phytase activity and zinc . . . 96

Plasma alkaline phosphatase activity and plasma
zinc, phosphorus, and calcium concentrations
in pigs receiving different dietary levels of
supplemental phytase activity and zinc . . 98

XII

Table

Table

Table

Table

Table

Table

Table

Table

Plasma iron, copper, and magnesium
concentrations in pigs receiving different
dietary levels of supplemental phytase
activity and zinc . . . . . . .

Balance of phosphorus, calcium, and zinc in
pigs fed the basal diet or basal diet

supplemented with zinc sulfate or microbial

phytase . . . . . . . . . .

'Plasma inorganic phosphorus, calcium, and

zinc concentrations and plasma alkaline
phosphatase activity of pigs fed the basal
diet or basal diet supplemented with zinc
sulfate or microbial phytase

in experiment 3.2 . . . . . . .

Composition of the basal and experimental
diets . . . . . . . . . . . . .

Probabilities of type I errors for test of
significance of main effects and
interactions on response measures .

Standard errors of mean differences and
degrees of freedom for various measures .

Daily gain, feed intake, and gain/feed of
pigs receiving different levels of
supplemental microbial phytase, vitamin D,
and calcium in the diets . . . . . .

Plasma concentrations of inorganic P and
Ca, and plasma alkaline phosphatase
activity of pigs receiving different
levels of supplemental microbial phytase,
vitamin D, and calcium in the diets .

XIII

100

102

.104

120

123

.124

125

128

LIST OF ABBREVIATIONS

ADFI average daily feed intake
ADG average daily gain

AP alkaline phosphatase

BD basal diet

PU phytase unit

XIV

INTRODUCTION

Over 60 to 70% of P in food of plant origin is bound
with myo-inositol phosphates as phytate that is poorly
available to simple-stomached animals such as pigs and
poultry. The low bioavailability of plant P to these animals
results in the need for addition of inorganic P to the diets
to meet their P requirements. Because inorganic P deposits
are limited, this supplementation not only increases feed
cost, but also sometimes causes feed P shortage, which was
the case in the 19705 in the USA. Moreover, the unutilized
dietary P excreted in the manure contributes to P pollution
in areas of intensive animal agriculture.

More than 20 years ago, attempts were made to use
phytase, an enzyme that hydrolyzes phytate, in the feed to
improve phytate-P utilization. However, because of high
anticipated phytase production cost compared to the
relatively low price of inorganic P sources, the potential
for phytase never received adequate attention. Recent
heightened environmental awareness of P and nitrogen
pollution originating from animal manure in some European

countries as well as in the USA has renewed the interest in

 

 

2
phytase. Modern biotechnology has facilitated the
development of commercial phytases. Currently, a limited
supply of microbial phytase is available in Europe, and
effects on phytate-P from plant feeds for pigs and poultry
have been examined. However, the data reported on pigs has
been mainly generated from digestion or balance trials with
single or double doses of phytase in the diets. The effects
of supplemental microbial phytase on P status in plasma and
performance of pigs were not studied.

This research was undertaken with weanling pigs
supplemented with a series of levels of microbial phytase in
typical corn-soybean meal diets. The objectives were to
determine the effectiveness, optimal dietary activity, and
inorganic P equivalents of supplemental phytase. In
addition, the improvement in dietary Ca and Zn utilization
by added phytase, and the effect of dietary concentrations
of Ca, Zn, and vitamin D on the efficacy of phytase were

determined.

LITERATURE REVIEW

Phosphorus

1. Phosphorus has versatile functions in animal body

 

Phosphorus accounts for approximately 1% of the body
weight of mature pigs (Peo, 1991). Although this is only 25%
of the total mineral in the body and ranks second to that of
Ca (Cromwell and Coffey, 1991), P is probably the most
protean of all the mineral elements. About 80% of P is found
as hydroxyapatite in the bones and teeth, so that P with Ca
plays vital roles in the development and maintenance of the
skeletal tissues (Underwood, 1981). The remaining 20% of P
is widely distributed in the fluids and soft tissues where
it functions in almost all processes and aspects of
metabolism (Peo, 1991). As a part of RNA and DNA, P is
essential for cell reproduction. As a part of organic
phosphate such as ATP, P is necessary for energy
transduction. As a part of phospholipid, P is essential for
maintaining membrane integrity and is involved in fat
transport. As an anion, P cooperates with other mineral
elements to maintain osmotic and acid-base balance. In

addition, P is involved in the control of appetite and in

4

the efficiency of feed utilization (Underwood, 1981).

Deprivation of dietary P results in an initial fall
in plasma inorganic P concentration (hypophosphatemia), a
rise in plasma Ca concentration (hypercalcemia), and a rise
in plasma alkaline phosphatase (AP) activity (Miller et al.,
1964; Underwood, 1981; Engstrom et al., 1985; Pointillart et
al., 1987; Pointillart, 1991). Hypophosphaturia,
hypercalciuria, and hyperhydroxyprolinuria often follow
(Pointillart et al., 1987; Nasi, 1990; Pointillart, 1991).
Renal 1-a1pha-hydroxy1ase and plasma 1, 25-(0H)g§ levels
are increased (Littledike and Goff, 1987). If sufficiently
severe or prolonged, clinical signs of P deficiency appear.
These include depressed appetite, poor rate and efficiency
of gain, rickets in the young and osteomalacia in the old,
and impaired fertility (Underwood, 1981, NRC, 1988).
However, these clinical signs are almost indistinguishable
from those resulting from deficiencies of dietary Ca or

vitamin D (Peo, 1991).

2. Phosphorus metabolism depends on Ca. vitamin D. and

 

other factors

Phosphorus is absorbed mainly in the proximal end of
the duodenum in the orthophosphate (PQf) form (Bartter,
1964; Irving, 1964), so that P absorption in the large
intestine of pigs appears negligible (Jongbloed, 1987).

Although the mechanism of P absorption is still unclear, the

5
factors influencing the amount of dietary P absorbed have
been elucidated (Peo, 1991). These factors include 1)
dietary level and source of P, 2) dietary Ca and vitamin D
level, 3) dietary ratio of available P and Ca, 4) intestinal
pH, and 5) other minerals that are antagonistic to P
absorption (Underwood, 1981; Peo, 1991). Normal dietary
levels of Ca enhance P absorption (Fox et al., 1978), but
high levels of dietary Ca generally decrease P absorption
(Jongbloed, 1987).

The P concentration of whole blood ranges from 35 to 45
mg/dL (Peo, 1991), while plasma inorganic P concentrations
are about 7 to 10 mg/dL (Miller et al., 1964; Ullrey et al.,
1967; Hancock et al., 1986; Miller and Ullrey, 1987:
Friendship and Henry, 1992). Low plasma P concentrations
result in poor performance, probably due to the impairment
of the extensive metabolic functions associated with P
(Hays, 1976). Phosphorus is excreted from the body via the
feces and urine (Moore and Tyler, 1955a, b; Pike and Brown,
1984). Thus, digestibility determinations are apparently
incomplete estimates of the biological value of a P
supplement (Peo, 1991). Various response criteria have been
used to determine dietary P availability by pigs. Peo (1991)
reviewed a number of studies conducted from 1962 to 1986 and
classified the sensitivity of the commonly used criteria as
1) high: physical traits of bone and percent bone ash (for

the young); 2) good: gain, feed conversion, and serum AP

6
activity; 3) low: serum Ca and P concentrations and percent
bone ash (for the adults); and 4) very low: percent

composition of bone ash.

3. Phosphorus supplements in diets are very costly

 

Corn-soybean meal diets for swine contain approximately
.3% of total P, which would be sufficient to meet the P
requirement of growing pigs if it was completely
bioavailable (Simons et al., 1990; Cromwell., 1991).
However, the average bioavailability of P in these diets is
only 15% (Cromwell and Coffey, 1991). Thus, supplements of
an inorganic P source (e.g. dicalcium phosphate or
defluorinated phosphate) or animal products (e. g. meat and
bone meal or fish meal) in the diets are necessary to
prevent deficiencies of P in pigs. Indeed, supplementation
with inorganic P effectively meets the P requirement of pigs
and thereby maximizes overall performance (NRC, 1988).

However, there are three major problems associated with
inorganic P supplementation. First of all, inorganic P is
quite expensive and ranks next to corn and soybean meal as
the major expense of swine diets. At least $2 or 3 for each
marketing pig would be saved if inorganic P supplementation
could be obviated (NAS, 1974). Next, inorganic P is a non-
renewable P resource. Continuing use of inorganic P
supplements at the current rate for animal production will

lead to exhaustion of these limited deposits (NAS, 1974).

7
Finally, and probably most important, part of the
supplemental inorganic P, and most of the dietary phytate-P
ends up in swine manure and results in excessive P entering
the environment (Cromwell and Coffey, 1991). Over 100
million tons of manure are excreted by livestock and poultry
in the United States annually, with approximately 18% from
swine and poultry (Sommers and Sutton, 1980; Gilbertson et
al., 1984). Because P concentration in non-ruminant manure
is twice as high as that in manure of ruminants, wastes of
swine and poultry contribute one-third of the total P
excreted in animal waste (Sommers and Sutton, 1980;
Gilbertson et al., 1984). The amount of P applied to soils
in animal waste, together with chemical fertilizer, often
exceeds the amount needed for plant growth. As a result, P
tends to accumulate in the soil and becomes a major
pollutant of the environment (Tunney, 1990; Lenis, 1989).
Phosphates move into surface water from surface run—off and
soil erosion and then stimulate growth of algae and other
aquatic plants there (Sharpley and Menzel, 1987). This
eutrophication decreases the quality of fresh water,
diminishes its oxygen content, and thereby creates an
undesirable environment for fish and other wildlife
(Cromwell and Coffey, 1991; Swick and Ivey, 1992). In the
Netherlands, Denmark, Belgium, and some parts of France,
pollution of the environment with P and nitrogen originating

from animal manure is becoming a major problem (Lenis,

8
1989). The same concern is also becoming an important issue
in the USA (Cromwell and Coffey, 1991; Swick and Ivey,

1992), and probably in other parts of the world as well.

Phytate

1. Phytate stores phosphate and energy in plant seeds

 

Phytic acid was discovered by Harting in 1855 (Reddy et
al., 1982). Through extensive studies by many generations,
it becomes acceptable that phytic acid is myo-inositol 1, 2,
3, 4, 5, 6-hexkis phosphate with an empirical formula of
C6H18024P6 (Reddy et al., 1982; Gibson and Ullah, 1990). In
plants, phytic acids exists as Ca-Mg—K salts called phytin
(Cosgrove, 1980). The structure and conformation of phytic
acid has been a subject of controversy (Reddy et al., 1982).
Weingartner and Erdman (1978) proposed a partially
dissociated Anderson-based structure for phytic acid that
might occur at neutral pH, which explains cation bindings of
phytic acid.

Phytate rapidly accumulates in seeds during the
ripening period (Nahapetian and Bassiri, 1975). Possible
physiological roles for phytic acid include its role as a
storage of phosphate, an energy source, an inhibitor of
dormancy, and as a mineral storage site (Gibson and Ullah,
1990). In addition, phytic acid prevents aflatoxin

production in soybean seeds by making Zn unavailable to the

9
mold (Gupta and Venkatasubramanian, 1975). Phytic acid has
been shown to form an Fe chelate that inhibits Fe-catalyzed
hydroxyl radical formation and lipid peroxidation, thus
could be used as a food antioxidant (Graf and Eaton, 1990;
Empson et al., 1991). Phytate may protect the seed against
oxidative damages during storage (Hernandez-Unzon and
Ortega-Delgado, 1989). Furthermore, phytic acid affects

cooking quality in legumes (Bhatty and Slinkard, 1989).

2. thta e represent§_mQ§t of phosphorus in cereals and

legumes
Concentrations of phytate and phytate-P in various

foods have been summarized by Nelson et al. (1968) and Reddy
et al.(1982). In general, the amount of phytate varies from
.50% to 1.89% in cereals, from .40% to 2.06% in legumes,
from 2.00% to 5.20% in oil seeds, and from .40% to 7.50% in
protein products (Reddy et al., 1982). In cereals such as
wheat, rice, barley and rye, the majority of phytate locates
near the outside of the seed coat, whereas in corn, phytate
concentrates in the germ (O'Dell et al., 1972b). In legumes,
phytate is distributed throughout the entire protein complex
of the seed (Reddy et al., 1982). The uneven distribution of
phytic acid in different morphological components or parts
of cereal and legume seeds allows milling to selectively
remove or reduce phytate contents in certain products, which

will be discussed later.

10
3. Phytate is fairly indigestible by simple-stomached
animals

Phytate is a nutrient source because it could provide
animals with P. However, the biological availability of
phytate-P in feed to ruminants greatly differs from that to
nonruminants. Due to the rumen microorganisms, cattle and
sheep hydrolyze naturally-occurring or pure phytate fairly
efficiently (Reid et al., 1947; Raun et al., 1956). Nelson
et al. (1976) studied the hydrolysis of natural phytate-P
from soybean meal, sorghum grains, and corn meal in calves
and steers. They found that the initial phytate hydrolysis
occurred in the rumen and was complete before the feed
reached the other parts of the digestive tract. Clark et al.
(1986) fed 30 Holstein cows with diets of 50% grain and 50%
of corn-silage through the first 18 wk of lactation. Their
results indicated that cows ingested approximately 40 g
phytate-P daily and hydrolyzed 98% of it to inorganic P. In
general, biological availability of dietary phytate-P to
ruminants are 50% or greater (Reddy et al., 1982).

In contrast, pigs can utilize only a small portion of
dietary phytate-P. The biological values of phytate-P in
four earlier studies ranged from 25% to 40% (Reddy et al.,
1982). Calvert et a1. (1978) demonstrated that phytate-P
from barley and corn was about 17% and 8% digestible,
respectively, for growing pigs when phytate in the diets was

the major source of P. Recently, Jongbloed et al. (1992)

11
reported that the typical corn-soybean meal diets for swine
contained approximately .33% of total P, of which 78.8% was
as phytate-bound P. They found that the apparent
digestibility of phytate was 21.5% for duodenal digesta and
12.9% for overall. Comparable availabilities of phytate-P
from similar corn-soybean basal or control diets have been
shown in other studies (Pointillart et al., 1984, 1985,
1987; Nasi, 1990; Simons et al., 1990; Pointillart, 1991).
These values generally agree with the estimates of Cromwell
and Coffey (1991).

Phytate-P from cereal grains and legumes is also poorly
available to poultry (Nelson, 1967; Reddy et al., 1982).
Gillis et a1. (1957) fed chicks and turkeys 32P-labeled Ca-
phytate and 32P-labeled monosodium orthophosphate and
measured the amount of radioactivity retained in the tibia.
They found that chicks and turkey used P from Ca-phytate
only 10% and less than 2%, respectively, as effectively as P
from monosodium orthophosphate. Employing the chromic oxide
balance method, Nelson (1976) demonstrated that 4- and 9-wk-
old chicks and laying hens (single comb white leghorn)
respectively, hydrolyzed 0, 3, and 8% of dietary phytate—P
from corn which was the only grain source. However, other
researchers have reported relatively high biological values
of phytate-P in the diets for poultry (Reddy et al., 1982).

The bioavailability of dietary phytate-P from plant

foods to rats appears in the same range as that to swine and

12
poultry (Moore and Veum, 1982, 1983; Williams and Taylor,
1985). In a limited number of studies with humans, the
bioavailability of dietary phytate-P from wheat bran varies
from 40% to 60% (Sandberg et al, 1982, 1986, 1987; Sandberg

and Andersson, 1988).

4. Phytate utilization varies with dietary factors and age
Species is not the only determining factor of

availability of dietary phytate-P. Dietary ingredients,
dietary concentrations of phytate, P, Ca, and vitamin D, and
age of animals also influence the utilization of phytate—P.
Replacing corn in part by wheat (Pointillart et al., 1984),
triticale (Pointillart et al., 1987), or rye bran
(Pointillart, 1991) in diets for growing pigs significantly
enhanced phytate-P utilization. Improvement in phytate
hydrolysis was also shown in chicks and laying hens by
substituting 50% of the corn with wheat in the diets
(Nelson, 1976). Ranhotra et al. (1974) found that the amount
of phytate hydrolyzed increased with elevated dietary
phytate levels in rats. In contrast, Pierce et al. (1977)
reported that increasing dietary phytate-P level from .30%
to .38% resulted in impairment in overall performance and
development in young pigs (11-14 kg). Moore and Veum (1982)
demonstrated that phytate-P was more available to rats given
diets with low inorganic-P than diets with supplements of P.

This adaptation by rats appeared to be related to enhanced

13
phytase or alkaline phosphatase synthesis by the
gastrointestinal microflora stimulated by the low level of P
in the digesta (Moore and Veum, 1983). Ballam et al. (1985)
fed 3-wk-old broiler chicks corn-soybean meal diets
containing varying amounts of Ca and P. They found that
increasing dietary inorganic P level from .12% to .45%
improved phytate hydrolysis in the absence of added Ca
(dietary Ca level: .09%). Where all diets contained 1.0% Ca,
the addition of all levels of inorganic P (up to .80%)
decreased phytate hydrolysis by chicks. It is believed that
calcium binds phytate in the gastrointestinal tract by
forming an insoluble Ca-phytate complex, and thus rendering
both Ca and P in the molecule largely unavailable to
absorption (Wise, 1983; Nelson and Kirby, 1987). When there
is a low Ca to phytate ratio in the diet, the majority of
the dietary phytate could be hydrolyzed (Ballam et al.,
1985) and metabolized (Nahapetian and Young, 1980). However,
high levels of Ca in the diets of rats (Taylor and Coleman,
1979; Nahapetian and Young, 1980) and poultry (Edwards and
Veltmann, 1983; Ballam et al., 1985; Sheideler and Sell,
1987) were consistently shown to decrease the availability
of phytate-P. Mohammed et al. (1991) fed 1-d-old male
broiler chicks either a control diet containing recommended
levels of P, Ca, and cholecalciferol or experimental diets
low in P and with variable levels of Ca (normal or low) and

cholecalciferol (normal or high). They concluded that

14
lowering of Ca or raising cholecalciferol alone improved
phytate-P utilization and simultaneously applying both in
low-P diets restored all variables to the levels for the
control diet. Pointillart et al. (1989) found that
increasing Ca level from .9% to 1.4% in diets containing
0.5% total P (all plant origin) intensified the harmful
effects of P deficiency secondary to phytate feeding in
pigs, but not via reduced phytate-P availability. Generally,
the proportion of dietary phytate-P hydrolyzed increases
with age in poultry (Peeler, 1972) and pigs (Newton et
al.,1983). However, the opposite occurred as hens progressed
through the egg laying period (Scheideler and Sell, 1987).
Likewise, Nelson and Kirby (1979) observed significant
decreases in phytate hydrolysis during each of the first 4-
wk periods in rats. They also noted that mature rats
hydrolyzed only half of the dietary phytate utilized by

weanling rats.

5. Phytate is an antinutritional factor
The ability of phytate to form complexes with metal
ions and proteins has been studied extensively (Wise, 1983,
Champagne et al., 1990). At neutral pH, phosphate groups of
phytate have either one or two negatively charged oxygen
atoms (Reddy et al., 1982). It is apparent that various
cations could strongly chelate between two phosphate groups

or weakly within a phosphate group (Erdman, 1979). Maddaiah

15
et a1. (1964) found the following decreasing order of
stability of metal-phytate complex: 2n” > cu” > Co“‘> Mn++ >
ca“. In the intestine, phytate is able to bind to protein
via divalent cation bridges (Wise, 1983). Thus, numerous
studies have proven that phytate in plant foods reduces
bioavailability of essential mineral elements (Oberleas,
1973; O'Dell, 1979, Reddy et al., 1982) and protein (Rojas
and Scott, 1969; Prattley and Stanley, 1982). Of great
importance in both human and animal nutrition is the adverse
effect of phytate on Zn utilization.

Tucker and Salmon (1955) first discovered Zn deficiency
in pigs fed a diet composed of corn and soybean meal. Later,
O'Dell and Savage (1960) found that the poor Zn availability
in the soy protein, and in other plant foods as well,
resulted from the formation of a complex between Zn and
phytate, which prevented Zn from absorption. Oberleas et al.
(1962, 1966) demonstrated the same adverse effect of phytate
on Zn metabolism in pigs and rats. More importantly,
Oberleas et a1. (1962) observed that the addition of Ca to
phytate-supplemented diets further reduced utilization of
dietary Zn, but high levels of dietary Ca had no effect on
Zn utilization in the absence of phytate. It has been
assumed that the high amount of Ca in common diets may
result in coprecipitation of trace metals with Ca-phytate,
and may break the phytate-protein complex in the intestine

(Wise, 1983).

16

It is well established that Zn from plant origin foods
is less available than from animal origin (Reddy et al.,
1982, Bobilya et al., 1991), thus pigs receiving corn-
soybean meal diets require higher dietary Zn level than
those consuming casein-glucose diets (Shanklin et al., 1968;
NRC, 1988). Newton et al. (1983) reported that incorporating
10% or 20% wheat bran in diets for growing pigs decreased Zn
absorption. Sandstead et al. (1990) showed that as little as
26 g of wheat bran added to the daily bread of men fed
omnivorous diets impaired retention of both Zn and Ca.
Direct demonstration of inhibitory effect of phytate in
humans on a stable isotope Zn tracer absorption and on a
radioactive Zn tracer retention was given by Turnland et a1.
(1984) and Sandstrom et al. (1987), respectively. These
findings provided the basis for the thesis that Zn
deficiency among humans in Middle Eastern countries was in
part caused by the high phytate contents of the breads that

are the staple food of the poor (Prasad et al., 1963).

6. Phytate contegtgiin foods can be reduced bv processing
Several methods are shown to reduce or remove
considerable amounts of phytate in cereals and legumes.
Kumar et al. (1978) reported that cooking decreased both
water- and acid-soluble phytate-P in green gram, conea, and
chickpea. de Boland et al. (1975) found that autoclaving

inositol hexaphosphate and isolated soy protein for 2 hr

17
resulted in substantial loss of phytate. As discussed
earlier, phytate is stored as a source of P for seed
germination in plants. Thus, germination reduces or
eliminates considerable amounts of phytate from the seeds or
grains (Reddy et al., 1982). In addition, fermentation,
soaking, and autolysis may also appreciably reduce phytate
contents of plant foods (Reddy et al., 1982). Phytate
contents in common bean seeds (Phaseolus vulgari§ L.)
substantially decrease after prolonged storage (Hernandez-
Unzon and Ortega-Delgado, 1989).

It has been mentioned in the above section that phytate
unequally distributes in different morphological components
or parts of cereal grains. Therefore, selective removal or
reducing of phytate contents in certain products by
mechanical processes is possible. Milling followed by germ
separation can remove 89% of the phytic acid in corn in
which phytate concentrates in germ (O'Dell et al., 1972).
Milling should also remove appreciable amounts of phytate
from wheat, barley, rice, rye, and triticale, those grains
have phytate in the surface layer (Reddy et al., 1982).

Various chemical methods have also been used to remove
phytate from soybeans (Reddy et al., 1982). All the
procedures are based on the different solubility of phytate
and protein depending on pH. However, neither this approach
nor other operations described above are economical on an

industrial scale.

Phytase

1. Phytase initiates phytate degradation
Phytases, myo-inositol hexaphosphate phosphohydrolases,

comprise a family of enzymes that catalyze the stepwise
removal of inorganic orthophosphate from phytate. In
addition, phytases hydrolyze a variety of natural and
synthetic phosphorylated substrates accepted by nonspecific
acid phosphatase as well (Gibson and Ullah, 1990). Thus,
phytases are also special kinds of phosphatase by nature.
Two types of phytases are recognized (Gibson and Ullah,
1990). One is called 3-phytase (E. C. 3. 1. 3. 8) which
initiates the removal of phosphate groups attached to
position 1 or 3 of myo-inositol. The other is called 6-
phytase (E. C. 3. 1. 3. 26) which first frees the phosphate
at 6-position (Nayini and Markakis, 1984). Both enzymes
eventually deesterify phytic acid completely. Phytases
characterized from microorganisms and the filamentous fungi
belong to 3-phytase. The seeds of higher plants typically
contain 6-phytase. However, in the majority of examples, the
type of phytase activity is not described because of the
difficulty in characterization and/or the existence of mixed
intermediate products (Gibson and Ullah, 1990). In general,
both types of activity are referred to as phytase. The unit
of phytase activity described in this thesis is defined as

the amount of enzyme that liberates 1 nmol of inorganic P

18

19
from sodium phytate per minute at pH 5 and 37°C. Different
units used in other studies are converted when discussed
herein.

Phytase activity from rice bran was one of the first
enzymes exhibiting phosphomonoesterase activity to be
characterized (Suzurk et al., 1907). But, the current best
characterized phytase is produced by a strain or variety of
Aspergillus niger, A. ficuum NRRL 3135. Soybean phytase has
also been well studied. Gibson and Ullah, with their
associates have been actively involved in the biochemical
characterization of these two phytases and the cloning of
the genes for these enzymes. Most of their work, plus that
of others, has been summarized (Gibson and Ullah, 1990).
From these data, we understand that A. ficuum phytase is a
3-phytase and a glycoprotein with a molecule weight between
85-100 KDa. Soybean phytase is a 6-phytase with a molecular
weight approximately 50 KDa. Both enzymes are most active
around pH 5.0, with the fungal enzyme having a broader pH
profile in the acidic range. Both possess optimal activity
at 55-58W3. Both are inhibited competitively by phosphate,
but soybean phytase is more sensitive to the inhibition. The
fungal phytase has much higher turnover number for phytic
acid than the soybean phytase. Other properties of these two
enzymes have been described by Gibson and Ullah (1990). The
enzymatic properties of other phytases from certain cereals

and legumes have also been studied (Reddy et al., 1982).

20

2. Phytase activity occurs in microorganisms, plants, and

animal tissues
Although phytase is formed during germination and may

play key roles in this process (Reddy et al., 1982), it is
also widely distributed in microorganisms and animal tissues
(Bitar and Reinhold, 1972; Cosgrove, 1980; Nayini and
Markakis, 1986). The exact numbers of microorganisms capable
of producing phytase is unknown. But it is known that many
microorganisms produce intracellular phytase (Cosgrove,
1980; Nayini and Markakis, 1986). Shieh and Ware (1968)
conducted a painstaking survey of more than 2000
microorganisms and observed that 30 of the isolates produced
extracellular phytase activity. All the extracellular
phytase producers were of fungal origin, among which A;
ficuum NRRL 3135 generated the highest amount of enzyme on a
corn-starch-based medium with limited inorganic P. Their
findings have been extremely important because A. ficuum or
its mutants has been extensively used as a rich source of
microbial phytase. Lopez et al. (1983) found that 16
bacteria strains isolated from a natural lactic fermentation
of corn meal had active phytase. Kang et al. (1988) isolated
67 strains from the fermenting corn and soybean meals with
phytase activity. They identified Bacillus lichenforms and
Enterobacter cloacae as the highest phytase producers.
Phytase activity was also found in culture filtrates of

Bacillus subtillis (Powar and Jangannathan, 1967). In

21
addition, phytase activity was also detected in a number of
soil microorganisms (Cosgrove, 1980), yeasts (Nayini and
Markakis, 1984; Lambrechts et al., 1992), molds used in
oriental food fermentation (Wang et al., 1980), and rumen
(Raun et al., 1956).

Phytase has been reported in a wide range of seeds of
higher plants containing phytic acid (Reddy et al., 1982;
Gibson and Ullah, 1990). Among the commonly used grains and
legumes, wheat (Ranhotra and Loewe, 1975), triticale
(Pointillart et al., 1987), and rye (Pointillart, 1991) are
relatively rich in phytase. These grains and their by-
products are used as sources of supplemental dietary phytase
in both animal and human diets, which will be discussed
later.

Phytase activity was detected in the intestinal mucosa
of rat, chicken, calf, and man (Bitar and Reinhold, 1972).
However, phytase activity in the intestinal mucosa of pigs
is negligible if it is really there (Pointillart et al.,
1984). Mucosal production of phytase is induced by low
dietary P in chicks (McCuaaig et al., 1972) but not in rats
(Moore and Veum, 1983). Zinc deficiency markedly reduces
production of endogenous phytase (Davies and Flett, 1978).
In summary, phytase in the gastrointestinal tract of animals
may originate from ingested plant ingredients, the gut
microflora, and endogenous production by the intestinal

mucosa. However, the total phytase activity in the

22
gastrointestinal tract of pigs fed ordinary corn-soybean

meal diets is negligible (Jongbloed et al., 1992).

3. Phytase releases phytate-phosphorus from plant food§:in

 

Kim

Incubating synthetic or natural phytate in phytase
activity bearing microorganism cultures results in
substantial hydrolysis of phytate. Raun et al. (1956) found
that washed suspensions of rumen microorganisms were able to
hydrolyze appreciable amounts of Ca-phytate as measured by
the presence of inorganic P following incubation in an
artificial rumen system. Lopez et al. (1983) found that
natural lactic acid fermentation of corn significantly
reduced phytate contents and thus increased the amount of
free P in the culture. Similar hydrolysis of phytate was
also seen in fermented peanut press cake (Fardiaz and
Markakis, 1981) and in fermented and germinated pearl millet
(Khetarpaul and Chauhan, 1990).

Nelson et al. (1968a) added A. ficuum phytase to a
liquid soybean meal and incubated the mixture for 2-4 h at
50 °C. After drying, the treated soybean meal was fed to 1-
d-old chicks. The birds utilized this hydrolyzed phytate-P
as efficiently as they did inorganic P. Under the same
light, Zhu et al. (1990) found that 81% of phytate-P was
converted to orthophosphate in a feed mixture of soybean

meal, corn and wheat bran when treated with native phytase

23
in wheat bran and ground corn by a 2-step process. The first
step consisted of two successive 2-h conditioning periods at
55°C for corn and soybean meal; one at pH 3.5 with citric
acid followed by another at pH 5.1. In the second, or
hydrolysis step, wheat bran (.25 parts), a rich source of
phytase, was mixed with the wet, citric acid-conditioned
corn and soybean meal, and the mixture was held for 8 h at
45°C and pH 5.1. After the enzymatic reaction, the increase
in orthophosphate accounted for 78% of the phytate-P
released and the mixture contained .42% inorganic P in dry
matter. A 17-d chick feeding trial showed that tibia bone
ash weights tended to be higher for chicks consuming the
treated, low-phytate feed than for those fed on the control
feed. Han and Wilfred (1988) applied crude culture filtrate
of A. ficuum to soybean and cottonseed meals for 24 h at
37°C. They found that 85% phytate in soybean meal and 67%
phytate in cottonseed meal was hydrolyzed. They also noted
that treatment at higher temperature (50 °C), pH 4-5.5, and
heating the substrates 1 h at 121 °C prior to the enzyme
treatment facilitated the hydrolysis of phytate by microbial
phytase. Simons et al. (1990) further characterized the in
vitro effects of A. ficuum phytase on degradation of phytate
in plant feeds. They demonstrated that the enzyme activity
maximized at both pH 5.5 and pH 2.5, and confirmed the
thermal stability of the enzyme resistant to heat of

pelleting previously shown (Jongbloed and Kemme, 1990).

24
Supplementing the microbial phytase at 1000 PU/g substrate,
they observed that hydrolysis of phytate in ground corn or
soybean meal was complete after an incubation of 1 h at 40
°C and was 80% in a corn-soybean meal diet for growing pigs
after 4 h at room temperature. In addition to orthophosphate
liberation, an increase in amount of protein in a solid
stable fermentation of canola meal by A. ficuum phytase was
also shown (Nair and Duvnjak, 1990). Based on the above
results, we can see the possibility of preparing low-phytate
diets for poultry and swine by treating plant feeds with
either microbial or cereal phytase in vitro. However, this
process seems uneconomical (Zhu et al., 1990). Han (1989)
estimated that feeding the phytase treated feed would be
approximately 17 times more expensive than the addition of
fertilizer-grade P, without crediting other benefits

associated with phytase application.

4. Phytase supplementation in diet§_improve§_phvtate-

phosphorus bioavailability to poultry

Attempts to add phytase to chick diets were dated back
to 1944, and supplements of sun-cured alfalfa meal, wheat
meal, barley meal, and lysed E. Coli. cellular material to
the diets promoted phytate-P utilization by chicks (Harms
and Damron, 1977). Nelson et a1. (1971) supplemented a corn—
soybean meal diet for chicks with preparations of A. ficuum

phytase. They found that the supplemental microbial phytase

25

was completely effective in hydrolyzing the dietary phytate-
P in the alimentary tract of the chick. Twenty years later,
the effectiveness of A. ficuum phytase in improving phytate-
P availability to poultry was consistently confirmed by a
number of researchers (Kiisken and Piironen, 1990; Saylor,
1991; Schoner et al., 1991; Simons et al., 1990; Swick and
Ivey, 1990). However, the amounts of dietary microbial
phytase needed to match the effect of recommended levels of
supplemental inorganic P in these studies were variable.

Simons et al. (1990) supplemented graded levels of A;
ficuum phytase in diets composed of corn, soybean meal,
sorghum meal, and sunflower meal for broilers. The results
indicated that supplementing phytase increased dietary P
availability to 60% and decreased the amount of P in the
dropping by 50%. Though phytase up to 1500 PU/g diet further
improved weight gain and P availability, phytase at 750 PU/g
diet resulted in body weight gain to be equivalent to that
obtained with diets supplemented with inorganic P. Schoner
et al. (1991) reported that supplementing the microbial
phytase at 800 PU/g diet resulted in a higher total P
retention than that of control diets with added inorganic P.
Moreover, Kiisken and Piironen (1990) reported that phytase
added to a barley-oat diet at 500 PU/g gave performance
equivalent to that of inorganic P supplemented diet. In
contrast, Saylor (1991) found that supplemental phytase at

1000 PU/g of a corn-soybean meal diet was insufficient to

26
release the phytate-P completely. Swick and Ivey (1990)
demonstrated that chicks supplemented with phytase at 450
PU/g had a 20% improvement in dietary P retention but a
slower gain, compared with that fed inorganic P. The
variations of phytase activity to maximize the utilization
of dietary phytate-P in these studies may have been
attributed to many factors (Swick amd Ivey, 1992). However,
differences in diet composition and P concentration could be
one of the major reasons. As previously discussed, dietary
phytase activity originating from ingredients, dietary Ca
and other nutrients affect phytate utilization and in turn
phytase efficacy. Improvements of phytate-P utilization was
linearly related to supplemental phytase at low-P levels
(Schoner et al., 1991). But, the improvements diminished
with increased inorganic P concentrations in the diets
(Swick and Ivey, 1991). The inorganic P equivalent of
phytase was proposed as 700 PU to 1.0 g P from dicalcium

phosphate (Schoner et al., 1991).

5. Phytase supplementation in diets improves phytate—

 

phosphorus bioavailabilitv to swine

Recently, two sources of phytase have been shown to
effectively improve phytate-P utilization in swine diets.
One is cereal phytase. Using corn-soybean meal diets as the
control, Pointillart and associates investigated the effects

of cereal phytase from wheat (Pointillart et al, 1984),

27
triticale (Pointillart et al., 1987), and rye bran
(Pointillart, 1991) on dietary phytate-P availability to
growing pigs. The actual determined phytase activity in the
experimental diet incorporated with 90% wheat, 80%
triticale, or 20% rye bran was 160, 440, and 1200 PU/g,
respectively. In contrast, the phytase activity in the
control corn-soybean meal diets was negligible (0 to 20
PU/g). The measured criteria included P and Ca balance, bone
and plasma concentrations of P and Ca, plasma vitamin D
metabolites and parathyroid hormone, bone bending moments
and intestinal phosphatase activities. The responses of
these measures consistently indicated that all three cereal
phytases significantly improved dietary phytate—P
utilization and reduced, as expected, fecal P excretion.
Meanwhile, dietary Ca utilization was also enhanced.
Besides, Newton et al. (1983) reported that adding 10% or
20% of wheat bran in the diets for growing pigs elevated
dietary P absorption.

The other source of phytase is microbial phytase,
almost exclusively from A. ficuum. Simons et al. (1990)
supplemented the enzyme at 1000 PU/g of a corn-soybean meal
diet and a practical Dutch diet for growing pigs. Similar
improvement in apparent P digestibility (24%) and reduction
in fecal P excretion (35%) was shown in both diets by
supplemental phytase. Nasi (1990) found that supplementing

the enzyme at 500 PU/g of corn-soybean meal diets improved P

28
digestibility to the same level of inorganic P supplemented
control diets. But, a larger proportion of dietary P was
retained in phytase-supplemented diets than in the control
diets, indicating that utilization of dietary P was improved
by supplemental phytase as well. Leunissen and Young (1992)
demonstrated that supplemental phytase at 500 PU/g diet for
weanling pigs increased the availability of dietary P
equivalent to that of adding .17% of inorganic P. They did
not show further benefit of supplemental phytase by doubling
the dose. However, the basal diet used by them contained, in
addition to corn and soybean meal, 17.0% canola meal, 10%
whey, and .04% calcium phosphate which resulted in a high
dietary P level (.55%). Cromwell (1991) and associates
supplemented corn-soybean meal diets for growing-finishing
pigs with the microbial phytase at 0, 500, and 1000 PU/g and
measured the responses of performance and bone traits. Based
on the results, Cromwell (1991) proposed that supplemental
phytase at 1000 PU/g diet would release sufficient available
P from phytate in corn-soybean meal diets to almost meet the
P requirement of finishing pigs (NRC, 1988).

In contrast, earlier attempts to feed Saccharomyces
cerevisiae or other phytase-containing yeast culture to pigs
failed to show improvements in phytate-P availability
(Cromwell and Stahly, 1978; Chapple et al., 1979; Shurson et
al., 1983). However, these researchers used phytase products

with undefined activity, and(or) used mainly weight gain as

29
the response measure. Thus, the ineffectiveness of yeast
phytase may have been attributed to a possible activity
insufficiency of the products, a possible incompatibility of
these enzymes with the low pH in the stomach of pigs, and a
possible incompleteness or insensitivity of the response
measures. The A. ficuum phytase resulting in positive
response in the above discussed trials was acid and heat
resistant over a broad pH and temperature (pH 2.0 to 6.0,
and up to GO‘Kn Simons et al., 1990). The effectiveness of
the enzyme in vivo in hydrolysis of phytate was confirmed by
Jongbloed et al. (1992). Using two simple T-cannulas in the
duodenum and terminal ileum of pigs, they demonstrated that
supplementing A. ficuum phytase at 1500 PU/g in the diets
degraded a substantial portion of dietary phytate in the

gastroduodenal section.

6. Phytase supplementation in diets improves phytate
degradation in humans

Dietary fiber is advocated increasingly for humans for
its purported health benefits. Increasing dietary
consumption of cereal fibers, legumes, and soy protein
isolates has become a trend and may result in an increased
intake of phytate. The major concern in human nutrition is
the adverse effect of phytate on mineral metabolism,
particularly on trace elements, rather than P

bioavailability (Reinhold et al., 1976; Davis, 1979; Wise,

30
1983; Sandstrom et al., 1987; Sandstead et al., 1990;
Sandstead, 1992). However, endogenous phytases in bran have
a major effect on phytate hydrolysis in humans and the
amount of activity varies with the method of processing of
the fiber (Sandberg et al., 1982, 1986, 1987; Sandberg and
Andersson, 1988). Sandberg et al. (1987) fed the raw or
extruded wheat bran to seven adult patients with ileostomies
and found a significant reduction in phytate digestibility
in the extruded bran compared to the unprocessed bran. The
deactivation treatment of the bran did not make it resistant
to hydrolysis, and the differences in phytate hydrolysis
between these types of bran were directly related to the

phytase activity (Sandberg and Andersson, 1988).

Summary

Phosphorus is the most protean mineral element in the
animal body. Supplementing inorganic P to a corn-soybean
meal diets for pigs and poultry is necessary but increases
feed cost and manure P load on the environment. Though the
structure and physiological roles of phytate in plants are
still unclear, distribution and bioavailability of phytate
in the commonly used cereal and legume foods are well
elucidated. Phytase can degrade phytate and the activity
occurs widely in microorganisms, plants, and animal tissues.

A. ficuum phytase is the currently best characterized and

31
extensively used. Supplemental microbial or cereal phytases
effectively hydrolyze phytate-P and thus improve its
bioavailability to simple-stomached animals. The
improvements have been shown both in vitro and in vivo. As
expected, animals supplemented with dietary phytase or fed
phytase-treated diets excrete less P in the manure. Besides,
utilization of dietary Ca was also improved by supplemental
phytase. Nevertheless, supplemental dietary phytase in swine

and poultry production still remains uneconomical.

EXPERIMENTAL SERIES I
LINEAR IMPROVEMENTS IN PHYTATE PHOSPHORUS
BIOAVAILABILITY BY SUPPLEMENTAL DIETARY PHYTASE

(Submitted to J. Anim. Sci.)

ABSTRACT

Two experiments were conducted with weanling pigs to
determine the effectiveness of a dietary supplement of A;
pigs; phytase in improving the availability of phytate-P in
corn-soybean meal diets without supplemental inorganic P.
Exp. 1.1 consisted of two P and Ca balance trials and two
feeding trials. Twelve pigs (8.18 i .44 kg BW) were housed
individually in stainless steel metabolism cages. Six pigs
received 750 or 687 phytase units (PU)/g of basal diet and
the other six pigs received the basal diet without
supplemental phytase as control. In Exp. 1.2, ninety-six
pigs (8.81 i .75 kg BW) were allotted to 16 partially-
slotted floor pens and were supplemented with 0, 250, 500 or
750 PU/g of basal diet for 4 wk. Individual pig weights and
pen feed consumption were measured weekly. Blood samples
were taken from all pigs at the end of each trial in Exp.
1.1 and from three pigs per pen weekly in Exp. 1.2 to
measure serum (plasma) inorganic P (P) and Ca
concentrations, and alkaline phosphatase (AP) activities.
The results of Exp. 1.1 indicated that dietary phytase
increased P retention by 50% (P < .0001) and decreased fecal

P excretion by 42% (P < .0001). Pigs receiving dietary

32

33
phytase had serum P and Ca concentrations, and serum AP
activities that were near normal, whereas control pigs had
values indicative of a moderate P deficiency. Favorable
effects of phytase disappeared when removed from the diet.
The results of Exp. 1.2 indicated a linear increase in
plasma P (P < .001), ADG (g < .07), and ADFI (g < .01) with
increased dietary phytase activity. Plasma AP activity
decreased linearly with increased dietary phytase activity
up to 500 PU/g of diet. Gain/feed and plasma Ca
concentration were measures least affected by dietary
phytase activity. In conclusion, supplements of A. niger
phytase up to 750 PU/g of feed in corn-soybean meal diets of
weanling pigs resulted in a linear improvement in
utilization of phytate-P.
Key words: Pigs, Phytase, Phosphorus, Plasma Ca, P, and

Alkaline Phosphatase

Introduction

More than 60% of P in corn and 50% of P in soybean meal
is in the form of phytate, which is poorly available to pigs
and other simple-stomached animals (Reddy et al.,1982).
Attempts have been made to use microbial phytases to improve
the availability of phytate-P in these feeds. Nelson et al.
(1971) fed day-old chicks with an acetone-dried preparation

of phytase from A. niger and demonstrated an improvement in

34
availability of phytate-P from corn and soybean meal.
Recently, positive effects of dietary supplemental phytase
produced by the same fungal species on phytate-P utilization
in broilers and pigs have been demonstrated by Simons et al.
(1990) and Nasi (1990). In contrast, feeding Saccharomyces
cerevisiae or other phytase-containing yeast cultures to
pigs failed to show improvements in phytate-P availability
(Cromwell and Stahly, 1978; Chapple et al., 1979; Shurson et
al., 1983). However, these researchers used phytase products
with undefined activity, and(or) used mainly weight gain as
the response measure. Therefore, the present two experiments
were conducted with weanling pigs to determine 1) the
efficacy of supplements of A. niger phytase to a
corn-soybean meal diet without supplemental inorganic P on P
and Ca balance, serum inorganic P (P) and Ca concentrations,
and alkaline phosphatase (AP) activity; 2) the possible
carry-over effects of phytase feeding on serum P and Ca
concentrations, and AP activity after phytase withdrawal;
and 3) the statistical relationship between supplemental
dietary phytase activity and the resultant improvement in
dietary phytate-P utilization as measured by performance and

plasma P status.

Materials and Methods

Phytase. The microbial phytase used in this study was
produced by A. niger (var. ficuum). The enzyme product was
kindly provided by Alko Ltd., Rajamaki, Finland and the
activity was approximately 500,000 phytase units (PU)/g (one
PU is defined as the amount of enzyme that liberates 1 nmol
of inorganic P from sodium phytate per minute at pH 5 and 37
°C). Actual phytase activity was confirmed by the assay
method of Han et al. (1987) before the product was mixed
with other feed ingredients in the preparation of the
complete diet.

Animals and Treatments. All pigs used in the two
experiments were 4-wk old crossbred (Landrace-Yorkshire-
Hampshire) weanling pigs. In Exp. 1.1, twelve pigs (8.18 i
.44 kg BW) were selected and allotted equally into two
groups receiving supplemental phytase (+ phytase) or no
supplemental phytase as control (- phytase). Pigs were
housed in individual stainless steel metabolism cages and
fed the low-P, basal diet (Table 1.1) for 2 wk to adjust and
deplete P reserves before the formal trials began. Four
consecutive trials were then conducted as follows: Trial 1,
first P and Ca balance for 7 d; Trial 2, free feeding for 10
d; Trial 3, second P and Ca balance for 7 d; and Trial 4,
free feeding basal diet for 14 d. Pigs in the + phytase

group received 750, 750, and 687 PU/g of basal diet in

35

36
Trials 1, 2, and 3, respectively. Supplemental phytase
activity in Trial 3 was reduced to approximately 90% of that
in Trials 1 and 2 based on the increase in age and in feed
intake of pigs. In Trial 4, these pigs were fed the same
basal diet as fed to the control group to determine if there
was any carry-over effect of phytase feeding on blood serum
P status.

In Exp. 1.2, ninety-six pigs (8.81 i .75 kg BW) were
split equally into heavy and light blocks based on body
weight. Within each block, 48 pigs were allotted to eight
pens with six pigs each. Four dietary levels of supplemental
phytase activity, 0, 250, 500, and 750 PU/g of basal diet
were assigned randomly to pens twice in each block and four
times in the whole experiment. Pigs were reared in
partially-slotted floor pens and given ad libitum access to
feed and water. Experimental housing was maintained at 22-25
0C, with a 12 h light:dark cycle. All pigs were fed the low
P, basal diet for 1 wk to deplete P reserves before the
formal trial.

Basal Diets. The basal diets were fortified corn-
soybean meal diets without supplemental inorganic P (Table
1.1). The diets provided adequate levels of all nutrients
(NRC, 1988) with the exception of P, Ca, and lysine in Exp.
1.1 and P and Ca in Exp. 1.2. Calcium carbonate was added to
provide a calculated Ca/P ratio of approximately 1.5 in the

basal diets. Concentrations of Ca and P in all experimental

37

Table 1.1. Composition and nutritive values of basal diets

Item Experiment 1.1 Experiment 1.2

Ingredient -------- g/kg --------
Corn (ground, shelled) 780.0 777.4
Soybean meal (44% CP) 200.0 200.0
Calcium carbonate (38% Ca) 10.0 10.0
L-Lysine HCl 2.6
Salt (NaCl) 3.5 3.5
Vitamin-trace mineral premix8 3.0 3.0
Vitamin E-Se premixb 3.0 3.0
Antibiotic premixc .5 .5

Calculated nutritive values (as fed)
ME, MJ/kg 13.8 13.8
CF, g/kg 155.0 155.0
Lysine, g/kg 7.8 10.2
Ca, g/kg 4.4 4.4
P, g/kg 3.2 3.2

a Supplied the following amounts per kilogram diet: vitamin

A, 1,980 IU; vitamin D3, 396 IU; menadione, 3.3 mg;

riboflavin, 2 mg; niac1n, 11 mg; d-pantothenic acid, 8 mg;

choline, 66 mg; vitamin Ba! 12 pg; Zn, 45 mg; Fe, 35 mg;

Mn, 20 mg; Cu, 6 mg; I, .12 mg.

b Supplied 10 IU of vitamin E and .2 mg of Se per kilogram
diet.

c Supplied 55 mg of chlortetracycline per kilogram diet.

38
diets were analyzed (Table 1.2). Synthetic lysine was not
incorporated into the diets in Exp. 1.1. Because we were
uncertain initially of the effectiveness of phytase in
releasing phytate-P from the basal diets, we considered that
it might be important to reduce dietary Ca and lysine
concentrations proportionately with the low P concentration
and to keep the ratios among these three nutrients close to
that of NRC (1988).

Table 1.2. Analyzed dietary Ca and P concentrations of
experimental dietsa

Item Phytase, Ca P
PU/g --- g/kg -----

Experiment 1.1

Trial 1 - Phytase 0 6.8 3.1

+ Phytase 750 5.8 3.2

Trial 2 - Phytase 0 6.8 3.1

+ Phytase 750 5.8 3.2

Trial 3 - Phytase 0 7.1 3.5

+ Phytase 687 5.8 3.3

Trial 4 0 8.9 3.7
Experiment 1.2

Diet 1, basal 0 5.0 2.8

Diet 2 250 5.5 3.2

Diet 3 500 5.0 3.1

Diet 4 750 5.0 3.3

a As fed basis.

39

Sample Collection and Measurements. In Exp. 1.1, P and
Ca balance trials were conducted as previously described
(Ilori et al., 1984). Collection period was 4 d and 3 d in
Trials 1 and 3, respectively. Feces of individual pigs were
collected daily and air-dried. Urine was collected into 2-L
plastic containers daily, and a 10% well-mixed sample was
stored at -20 0C for P and Ca analyses. At the end of each
trial, including the beginning and the end of the first wk
of Trial 4, blood samples were taken from each pig and serum
was prepared for assay of P and Ca concentrations, and serum
AP activity. Body weight also was recorded at each bleeding.
In Exp. 1.2, individual pig weights and pen feed consumption
were measured weekly. Blood samples were taken weekly from
three pigs per pen for assay of plasma P and Ca
concentrations, and plasma AP activity.

Assays. Concentrations of P in feed, feces, urine, and
blood serum or plasma were determined by a colorimetric
method (Gomori, 1942), and Ca concentrations were determined
by flame atomic absorption spectrophotometry
(Instrumentation Laboratory, Inc., Model IL 951). Serum or
plasma AP activity was determined on the day that blood
samples were drawn by the method outlined by Sigma Chemical
(1987).

Statistics. Differences in blood serum measures and ADG
between pigs fed diets supplemented with or without phytase

in Exp. 1.1 were analyzed statistically by simple p-test

40
rather than time repeated measurement because of the switch
in feeding method and the difference in supplemental phytase
activity in the diets between different trials. However, P
and Ca balance data in Trials 1 and 3 were pooled and
analyzed by time repeated measurements because percentage of
improvement and significance level of the improvement by
supplemental phytase in the two trials were almost
identical. There was also no interaction of trial (time) by
treatment. The results of Exp. 1.2 were analyzed as a
randomized complete block design with 4 treatments (4
phytase levels) in 2 blocks (heavy and light) with time
repeated measurements and the pen was the experimental unit.
Orthogonal polynomials of dietary phytase activity with
different measures were developed by procedures outlined by
Gill (1978). Bonferroni p-test was used for treatment mean
comparisons. Significance level was set as P < .05 unless
indicated otherwise. All analyses were conducted with the

SAS program (SAS, 1988).

Results

Experiment 1.1

Balance of P and Ca. The pooled P and Ca balance data
in the two trials are presented in Table 1.3. With similar
daily P intake, pigs fed phytase retained 50% more P daily

(2 < .0001) than control pigs. Meanwhile, daily fecal P

41
output was reduced by 42% (g < .0001) in pigs fed phytase.
Daily urinary P loss of pigs was extremely small compared
with their fecal P loss regardless of dietary phytase
supplementation. This resulted in an almost complete
retention of absorbed P in both control and phytase-fed pigs
and an almost identical increase of 23 percentage units (2 <
.0001) in apparent digestibility of P and percentage of P
retained/intake in pigs fed phytase.

Daily Ca intake of the control pigs was 14% higher (g_<
.01) than that of pigs fed phytase, due to the somewhat
higher Ca concentration of the control diet. But, neither
daily Ca absorbed nor daily Ca retained in control pigs was
increased. In contrast, pigs fed phytase retained slightly
more Ca and had 13 (g < .0001) and 14 (P < .02) percentage
unit increases in apparent digestibility of Ca and
percentage of Ca retained/intake, respectively. Daily fecal
Ca output in pigs fed phytase was reduced by 52% (P < .0001)
whereas daily urinary Ca output was not different (2_< .33)
from that of control pigs. In addition, fecal P and Ca
concentrations in pigs fed phytase were reduced by 45% (1.1
vs 2.0%, g < .0001) and 50% (1.1 vs 2.2%, P < .0001),

respectively.

42

Table 1.3. Balance of P and Ca in pigs fed diet with or
without supplemental microbial phytasea

- Phytase + Phytase SEDb P <
---------- Phosphorus, mg/d -------------
Intake 1,801 1,809 95 .93
Fecal 966 561 67 .0001
Urinary 4.0 3.5 1.0 .64
Absorbed 835 1,248 84 .0001
% Of intake 46.4 69.0 2.9 .0001
Retained 831 1,245 84 .0001
% of intake 46.2 68.8 2.9 .0001
% of absorbed 99.5 99.7 .2 .09
---------- Calcium, mg/d -------------
Intake 3,753 3,214 182 .014
Fecal 1,092 526 79 .0001
Urinary 615 460 153 .33
Absorbed 2,661 2,688 172 .88
% Of intake 70.9 83.6 2.0 .0001
Retained 2,046 2,228 250 .48
% of intake 54.5 69.3 5.1 .02
% of absorbed 76.9 82.9 5 8 .32

a Data presented here were pooled from the two balance
trials.

b Standard error of difference of two means (df of error,
10).

43

Serum Inorganic P and Ca Concentrations. and Alkaline
Phosphatase Activity. Effects of supplemental dietary
phytase on serum P and Ca concentrations, and AP activity
are summarized in Table 1.4. The initial concentrations of
serum P in pigs of the two treatment groups were essentially
the same. However, serum P concentrations in pigs fed
phytase increased to 7.0 mg/dL at the end of Trial 1 and
stayed above this level in Trials 2 and 3. In contrast,
serum P concentration in control pigs initially increased,
but then gradually decreased from 6.6 mg/dL at the end of
Trial 1 to 4.5 mg/dL at the end of Trial 3. The difference
in serum-P between the two treatment groups was 2.3 mg/dL at
the end of Trial 2 (P_< .0007) and 3.0 mg/dL at the end of
Trial 3 (2_<.0001). Serum Ca concentration of pigs fed
phytase was lower (P_< .001) than that of the control pigs
within these three trials. Serum AP activity of pigs fed
phytase was lower (g_< .03) than that of control pigs at the
end of Trial 3.

The significant differences in serum P and Ca
concentrations, and AP activity between control and phytase-
fed pigs disappeared in Trial 4 when phytase-fed pigs were
switched to the same low—P, basal diet as control pigs.
Serum P concentrations in the previously phytase-treated
pigs decreased and serum Ca increased to the levels of

control pigs. Serum AP activity was also similar to that of

44

Table 1.4. Serum inorganic P and Ca concentrations, and
serum alkaline phosphatase activity of pigs fed diet with or
without supplemental microbial phytase in experiment 1.1

Trial - Phytase + Phytase SEDa 2 <

---------- Serum inorganic P, mg/dL ------—----
Initial 5.4 5.2 .54 .84
Trial 1 6.6 7.0 .33 .22
Trial 2 5.1 7.4 .48 .0007
Trial 3 4.5 7.5 .48 .0001
Trial 4, wk 1 5.7 5.5 .41 .72

wk 2 5.8 5.7 .40 .94

------------ Serum Ca, mg/dL -----------------

Initial 12.3 12.9 .24 .05
Trial 1 12.3 11.0 .30 .001
Trial 2 13.9 10.8 .41 .0001
Trial 3 14.3 11.8 .33 .0001
Trial 4, Wk 1 17.0 16.2 1.00 .43

wk 2 14.8 15.0 .83 .86

------ Serum alkaline phosphatase, Ub/dL ----—---
Initial 22.3 23.4 4.03 .78
Trial 1 25.1 22.5 2.24 .27
Trial 2 18.1 16.8 1.54 .44
Trial 3 22.5 16.5 2.37 .03
Trial 4, wk 1 17.4 15.4 2.44 .45

Wk 2 17.0 15.9 3.07 .74

a Standard error of difference of two means (df of error,

10).

bOne unit of activity is defined as that amount of enzyme

that produces 1 umole of P-nitrophenol per minute under the
conditions of the assay (Sigma Procedure No. 425,

1987).

45

control pigs after phytase was removed from the diet, but it
did not change as much as the concentrations of serum P and
Ca. Compared with that at the end of Trial 3, serum AP
activity and serum P concentration in control pigs in Trial
4 decreased approximately 5 units (U)/dL and increased 1
mg/dL, respectively. These changes may have been due to the
switch from restricted feeding in Trial 3 to free feeding in
Trial 4 and an increase in age.

Weight Gain. Pigs fed phytase had a greater ADG (2 <
.06) than control pigs in Trial 2, when all pigs were
allowed to consume their diets on an ad libitum basis. The
ADG of pigs and the difference between control and treated
groups was relatively small in the two balance trials. In
the final week of Trial 4, control pigs grew faster than
pigs previously fed phytase, probably due to compensatory
growth. However, the ADG of pigs was really measured only to
indicate that pigs in both groups were in good health and
were gaining weight in the experiment (Table 1.5).

Table 1.5. Daily gains of pigs fed diet with or without
supplemental microbial phytase in experiment 1.1

Trial - Phytase + Phytase SEDa P <
Initial 65 71 20 .80
Trial 1 205 219 18 .48
Trial 2 370 436 32 .06
Trial 3 212 252 35 .28
Trial 4, wk 1 338 354 57 .79

Wk 2 411 275 101 .21

8Standard error of difference of two means (df of error,
10), unit = gram.

46

Experiment 1.2

glsspa Inorganic P and Ca Concentrations, and Alkaline
Phosphatase Activitv. Effects of dietary phytase activity on
plasma P and Ca concentrations, and AP activity are
presented in Table 1.6. Plasma P concentrations increased
linearly with increase in dietary phytase activity. The
responses could be represented by the following four

orthogonal polynomials:

Wk 1. Y = 4.10 + .00292X (E_< .001, £_= .97);
Wk 2. Y = 4.02 + .00322X (£_< .001, §_= .98);
Wk 3. Y = 3.69 + .00377X (B < .001, £_= .96):
Wk 4. Y = 3.23 + .00346X (2_< .001, §_= .99);

Where X = dietary phytase activity, PU/g; Y = plasma P
concentration, mg/dL. There was no maximum break point of
plasma P concentrations among the three phytase-supplemented
groups of pigs. Compared with suggested normal values (7 to
8 mg/dL, Ullrey et al., 1968), plasma P concentrations of
pigs receiving no phytase were less than 50% of norm and
pigs receiving the highest phytase activity (750 PU/g) were
slightly below the norm.

Plasma AP activity of pigs receiving no supplemental
phytase increased from wk 1 to wk 4 and eventually exceeded
(2 < .05) that of the three groups of pigs receiving
supplemental phytase at wk 4. Plasma AP activity decreased

linearly (P < .06) with increase in dietary phytase activity

47

Table 1.6. Plasma inorganic P and Ca concentrations, and
plasma alkaline phosphatase activity of pigs receiving
graded dietary levels of supplemental microbial phytase
activity in experiment 1.2

------ Phytase, PU/g of diet ------
Time 0 250 500 750
---------- Plasma inorganic P, mg/dL -------------
Initial 5.2 5.0 5.4 5.6
Wk 1 4.1“ 5.0x 5.2x 6.5V
Wk 2 3.9" 5.1x 5.5x 6.4y
Wk 3 3.4" 5.1x 5.4x 6.5V
Wk 4 3.2" 4.1x 5.0V 5.92
(SED°==.31, df of error = 45)
----------------- Plasma Ca, mg/dL --------------
Initial 11.6 12.2 12.0 11.6
Wk 1 12.3 12.4 12.5 11.9
Wk 2 13.0 12.6 12.4 12.4
Wk 3 12.6 12.2 12.3 11.8
Wk 4 12.6 12.4 12.2 11.9
(SED = .42, df of error = 28)
---------- Plasma alkaline phosphatase, Ub/dL ---
Initial 18.4 20.7 16.6 18.8
Wk 1 16.6 20.6 16.6 18.1
Wk 2 20.7 21.2 18.9 17.4
Wk 3 21.3" 20.8“‘ 16.6“x 16.0x
Wk 4 26.9" 19.7x 15.7x 16.6x

(SED 1.96, df of error = 48)
a Standard error of difference of means between any two
dietary levels of phytase activity at a given wk.
See Table 4 for enzyme unit definition.
‘“*~V" Means within a row lacking a common superscript
letter differ (P_< .05).

48
from 0 to 750 PU/g of feed at wk 3 but only up to 500 PU/g
of feed at wk 4. The relationships between plasma AP
activity (Y, U/dL) and dietary phytase activity (X, PU/g)
fit the following orthogonal polynomials:

Wk 3. Y

21.68 - .0008X (g < .06, r = .94);
Wk 4 Y = 26.94 - .0383x + .0000326X2 (2_< .05, R2 = .99).

There was no effect of dietary phytase activity on
plasma Ca concentration of pigs.

Wsight Gain. Fssd IntakeI and GainZFeed. Effects of
dietary phytase activity on ADG, ADFI, and gain/feed are
presented in Table 1.7. Pigs receiving supplemental phytase
grew faster than pigs receiving no phytase, and differences
were significant at wk 3 and 4. There was no significant
difference in ADG among the three groups of pigs fed
phytase, but a linear increase in ADG with increase in
dietary phytase activity was evident. The relationships
between ADG (Y, g/d) in wk 4 and over the entire period and
dietary phytase activity (X, PU/g) can be described by the
following orthogonal polynomials:

Wk 4. Y = 358 + .159x (P_< .06, r_= .98);

Overall. Y = 327 + .112X (P < .07, p_= .95).

Feed intake (Y, g/d) increased linearly as dietary
phytase activity (X, PU/g) increased and the responses at wk
3 and wk 4 can be described by the following orthogonal
polynomials:

Wk 3. Y = 867 + .253X (E < .01, §_= .95);

49

Table 1.7. Daily gain, feed intake, and gain/feed of pigs
receiving graded dietary levels of supplemental microbial
phytase activity in experiment 1.2

------ Phytase, PU/g of diet ------
Time 0 250 500 750

---------- Daily gain, g/d -------------

Initial 108 139 133 156
Wk 1 242 295 307 294
Wk 2 300 326 365 365
Wk 3 371x 452V 436xy 462V
Wk 4 351x 411“’ 433y 475V
Overalla 316x 371“’ 385V 405y

(SEDb== 27 at a given wk and 22 for overall,
df of error = 46)

------------- Feed intake, g/d -----------

Wk 1 557 666 679 667
Wk 2 762 784 842 835
Wk 3 872x 985xy 984’” 1,084Y
Wk 4 992x 1,122x 1,144xy 1,292y
Overall‘ 796x 890xy 912“’ 961y

(SED = 54 at a given wk and 47 for overall,
df of error = 21)

----------- Gain/feed, g/kg ----—-----------

Wk 1 433 435 448 458
Wk 2 390 423 433 435
Wk 3 424 463 443 433
Wk 4 355 368 380 383
Overall 403 418 420 425

(SED = 28 at a given wk and 15 for overall,
df of error = 48)
3 Overall mean from wk 1 to wk 4.
b Standard error of difference of means between any two
dietary levels of phytase activity.
° Comparisons of overall means may not be used because of an
interaction (P<.001) of time by treatment on ADFI.
“'Y Means within a row lacking a common superscript letter
differ (g_< .05).
Wk 4. Y = 999 + .369X (P_< .0001, p_= .97).

50

Overall ADFI also responded linearly to the increase in
dietary phytase activity: Y = 812 + .208X (g_< .01, p_=
.97). However, this equation should be interpreted with
caution because of the interaction (2_< .001) of treatment
with time on ADFI.

Gain/feed appeared improved by the increase in dietary
phytase level, but the effect was nonsignificant.

For all the measures taken in Exp. 1.2, there was no
interaction between dietary phytase activity and block
(weight). Consequently, block effect was not included in

Tables 1.6 and 1.7.

Discussion

Results of the balance studies strongly indicate that
addition of A. niger phytase at 750 PU/g of corn-soybean
meal diet improved phytate-P utilization in weanling pigs.
Given similar daily intake of P, pigs receiving supplemental
phytase had a 23 percentage unit increase (2_< .0001) in
apparent digestibility of P than pigs receiving no
supplemental phytase. Correspondingly, daily fecal P
excretion was reduced by 42% (2_< .0001) in these pigs.
Simons et al. (1990) have reported that addition of Ay_pigsp
phytase at 1000 PU/g feed (phytase units
are converted to the unit as defined in our study when other

studies are discussed) to diets of growing pigs (35 to 70

51
kg) increased apparent digestibility of P by 24 percentage
units and decreased the amount of P in the feces by 35%.
Nasi (1990) used the same source of phytase as in this study
at 500 PU/g of corn-soybean meal diet for 95-kg pigs and
obtained an improved apparent digestibility of P that
increased to the same level as an inorganic P supplemented
diet and was 24 percentage units higher than that of diet
unsupplemented with phytase. Leunissen and Young (1992) also
demonstrated that supplements of the same phytase as used in
this study at 500 PU/g of diet for weanling pigs improved
apparent digestibility of P to the same level as their
positive control diet. However, the improvement over the
diet unsupplemented with phytase was 11 percentage units and
accounted for only 50% of that obtained in studies of Nasi
(1990) and Simon et al. (1990), and in this study. The low
improvement may be explained by the relatively high dietary
concentration of total P (.55% with .04% calcium phosphate
supplementation) and high apparent digestibility of P in the
basal diet (60%). In addition, both Nasi (1990) and we have
shown the same amount of increase in percentage of P
retained/intake as that of apparent digestibility of P. An
increase of 10 percentage units in the percentage of P
retained/absorbed due to 500 PU/g of diet has been found in
the study of Nasi (1990). A marginal effect of phytase on P
retained/absorbed was also demonstrated in this study, but

the improvement may be too small to consider. Phytase has

52
also been shown to improve bone strength and P concentration
(Cromwell et al., 1991; Leunissen and Young, 1992).
Therefore, A. niger phytase appears to improve phytate-P
utilization as well as digestion.

Supplemental microbial phytase seems to improve Ca
utilization in addition to P utilization as apparent
digestibility of Ca and percentage of Ca retained/intake
were both increased by 13 percentage units in pigs fed
supplemental phytase. However, this improvement may be
confounded with an effect of the lower daily Ca intake.
Nevertheless, comparable improvement in digestion and
utilization of Ca by dietary phytase has been previously
demonstrated (Nasi, 1990; Simons et al., 1990). Dietary
phytase may improve Ca utilization indirectly by improving P
utilization because dietary Ca will be well utilized for
skeletal growth only as dietary P is simultaneously
utilized.

The effectiveness of phytase in improving phytate-P
availability has also been shown by the responses in
performance and measures of blood P status. In Exp. 1.1,
pigs receiving supplemental phytase maintained serum P and
Ca concentrations, and serum AP activity near to the normal
range (Miller et al., 1964; Ullrey et al., 1967) whereas
pigs receiving no supplemental phytase developed a moderate
P deficiency. In Exp. 1.2, plasma P concentration, ADG, and

ADFI increased linearly as dietary phytase activity

53
increased. More convincingly, favorable effects of dietary
phytase on serum P and Ca concentrations, and AP activity in
pigs fed phytase in the first 3 trials of Exp. 1.1
disappeared when phytase was withdrawn from the diet. This
rapid and consistent change not only indicates no carry-over
effect of phytase feeding on the measures of blood P status,
but also confirms the effectiveness of phytase on phytate P
utilization. The ineffectiveness of yeast phytase on phytate
P availability in pigs (Cromwell and Stahly, 1978; Chapple
et al., 1979; Shurson et al., 1983) may be attributed to 1)
the possibility that activity of the yeast phytase products
may have been insufficient to produce a response, and (or)
2) a possible incompatibility of these yeast phytases with
the low pH in the stomach of pigs. The A. niger phytases
used in this study and by other researchers who obtained
positive results (Nasi, 1990; Simons et al., 1990; Cromwell
et al., 1991, Leunissen and Young, 1992) are acid and heat
resistant over a broad pH and temperature range (pH 2.0 to
6.0, and up to 60 °C). Therefore, we may reasonably expect
these enzymes to be active and to function in the stomach of
pigs under physiological conditions. This hypothesis has
been confirmed by Jongbloed et al. (1992). Using two simple
T-cannulas in the duodenum and terminal ileum of pigs, they
demonstrated that lowering of dietary A. niger phytase
activity (1500 PU/g) by 415 PU after gastroduodenal

degradation resulted in an increase in phytic acid

54
degradation of 70 percentage units. Subsequently, liberated
ortho-phosphates were absorbed in the small intestine, and
fecal P excretion was greatly reduced. Hence, supplemental
phytase may not only improve utilization of phytate-P in
cereal-plant protein diets, but also alleviate or eliminate
P pollution by reducing P in swine manure applied to the
land, a severe problem facing the swine industry today
(Cromwell, 1991).

Plasma P concentration seems to be the most sensitive
and convenient measure of dietary phytase on phytate-P
utilization in this study. Supplements of dietary phytase at
750 PU/g of feed produced comparable improvement in P
concentrations and AP activities in serum of pigs in Exp.
1.1 and in plasma of pigs in Exp. 1.2. However, significant
differences in serum Ca concentrations between pigs
receiving no phytase and 750 PU/g of diet shown in Exp. 1.1
was not seen in plasma Ca concentrations in Exp. 1.2. This
inconsistency may be partly accounted for by the differences
in dietary Ca concentrations in the two studies.

The linear effects of dietary phytase activity up to
750 PU/g of diet on most of the measures of phytate-P
utilization taken in this study were very consistent. This
was reflected by the extremely high repeatability of the
strong correlations and significance shown in the developed
orthogonal polynomials. Similar response patterns in chicks

to supplemental dietary phytase activity have been reported

55
by Nelson et al. (1971) and Schoner et al. (1991). However,
phytase dose-related responses in pigs appear to vary in
different studies. A linear relationship (2 < .01) between
supplementing dietary phytase at 0, 500, and 1000 PU/g of
diet and ADG and bone strength in growing pigs was shown in
one study (Cromwell, 1991), but the higher level of
supplemental dietary phytase (1000 PU/g) was more effective
in improving only bone strength in another study (Cromwell
et al., 1991). Similarly, there was no further beneficial
effect on any of the response criteria by doubling
supplemental phytase activity from 500 to 1,000 PU/g of diet
in weanling pigs in the study of Leunissen and Young (1992).
In comparison to the results of this study, the lack of
further improvement in phytate-P utilization by dietary
phytase activity higher than 500 PU/g of diet in the studies
discussed above may be explained by 1) the growing-finishing
pigs used by Cromwell et al. (1991) had a lower P
requirement than the weanling pigs we used; and 2) the basal
diet used by Leunissen and Young (1992) had a higher total
and available P concentration than our basal diets, due to
.04% supplemented calcium phosphate and different feed
ingredients, as mentioned above. Lower P requirement and
higher dietary available P concentration would lessen the
amount of phytate-P needed to meet the requirement of P in
pigs, thereby making the higher dietary phytase activity

unnecessary to release just this portion of P from phytate.

56
Cromwell et al. (1991) did not observe any effect of phytase
supplemented in the diets with adequate inorganic P. In
addition, inorganic P is a strong inhibitor of phytase in
vitro (Shieh and Ware, 1968; Gibson and Ullah, 1988).

Among all the measures made in this study, it seems
that only the response of plasma AP activity was maximized
at wk 4 of Exp. 1.2 at dietary phytase activity of 587 PU/g
of diet. However, this level of phytase activity may not be
the optimal dose that could maximize the improvement of
phytate-P utilization because other response measures still
showed linear increases up to 750 PU/g of diet. Even 750
PU/g of diet may still be inadequate for pigs to positively
maintain normal plasma P concentrations (Ullrey et al.,
1967). If plasma P concentrations are to be maintained at
approximately 7.5 mg/dL, pigs may need supplemental phytase
activity of 1200 PU/g of diet based on the orthogonal
polynomials developed. Therefore, higher dietary levels of
phytase activity should be tested in future studies to
determine the phytase activity at which response measures

are maximum.

Implications

The A. niger phytase used in this study was very effective
in improving the availability of phytate-P in a corn-soybean
meal diet for weanling pigs and may greatly reduce the need
for supplemental inorganic P, the third largest expense in
cereal grain-plant protein swine diets. Supplementary
phytase up to 750 units per gram of feed resulted in a
linear improvement in phytate-P utilization as measured by
plasma inorganic P concentration, average daily gain and
feed intake. Furthermore, supplements of 750 phytase units
per gram of feed increased absorption and retention of P by
23 percentage units and reduced fecal P excretion by 42%.
Higher levels of dietary phytase activity may be needed to
maximize this improvement and to eliminate the need for

dietary inorganic P supplementation for weanling pigs.

57

EXPERIMENTAL SERIES II
MAXIMAL IMPROVEMENTS IN PHYTATE PHOSPHORUS
BIOAVAILABILITY BY SUPPLEMENTAL DIETARY PHYTASE

(Submitted to J. Anim. Sci.)

ABSTRACT

Two experiments were conducted with crossbred weanling
pigs to determine the optimal dietary supplement of A. niger
phytase activity to maximize utilization of phytate-P in a
corn-soybean meal basal diet (BD) without added inorganic P.
In addition, the inorganic P equivalent of phytase
supplementation was determined. In Exp. 2.1, fifty pigs
(7.61 i .56 kg BW) were allotted to 10 pens and were
supplemented with 750, 1,050, 1,250, or 1,350 phytase units
(PU)/g BD, or were fed the BD plus .21% P as mono-dibasic
calcium phosphate (MDCaP) for 4 wk. In Exp. 2.2, twelve pigs
(6.39 i .74 kg BW) were individually housed in metabolism
cages and received BD, BD plus the optimal phytase activity
(1,200 PU/g), or BD plus .21% P as MDCaP for 2 wk. Weekly
measures included ADG, ADFI, plasma concentrations of P
(inorganic), Ca, Zn, Mg, Cu, and Fe and plasma alkaline
phosphatase (AP) activity in 3 pigs per pen in Exp. 2.1 and
in all pigs in Exp. 2.2. Feces and urine were collected from
all pigs individually for 4 d at the end of Exp. 2.2 to
determine P (total) and Ca balances. No improvements (2 >
.05) in ADG, ADFI, gain/feed or plasma AP activity beyond

1,050 PU/g BD were seen in Exp. 2.1. Quadratic relationships

58

59
between dietary phytase activity and these measures
consistently predicted maximum breakpoints at approximately
1,200 PU/g. Estimated maximum responses of these measures
were 90% or more of those in pigs receiving MDCaP. In
addition, 1,250 PU/g appeared adequate to maintain plasma P
and Ca concentrations in the normal range. Effects of
dietary phytase activity on plasma Mg, Cu, Fe, and Zn
concentrations were not significant. In Exp. 2.2, pigs
receiving 1,200 PU/g utilized dietary P more effectively (2
< .05) than pigs fed the BD or the BD plus MDCaP. Although
consuming 44% less P per day, these pigs retained only 7%
less P (g > .05) than pigs receiving MDCaP. One thousand
units of phytase activity supported retention of 1.1 mg P
from the BD and were equivalent in effect to .91 mg P from
MDCaP. Concentrations of plasma P were lower and plasma Zn
were higher in pigs fed 1,200 PU than in pigs fed MDCaP, but
other measures were not different between these two groups.
Supplements of A. niger phytase at 1,200 PU/g of a corn-
soybean meal diet for weanling pigs appeared to maximize
utilization of phytate-P and obviate the need for almost all
of an inorganic P supplement.
Key words: Pigs, Phytase, Phytate, Phosphorus, Plasma Ca, P,

and Alkaline Phosphatase

Introduction

The effectiveness of 5; pigs; phytase in improving the
utilization of phytate-P in corn-soybean meal diets for
weanling pigs has been shown previously (Lei et al., 1991;
Leunissen and Young, 1992). The effect was linear over a
range of dietary phytase activity from 0 to 750 phytase
units (PU)/g of corn-soybean meal diet (Lei et al., 1992a).
However, the relationship between dietary phytase activity
beyond 750 PU/g and further improvements in phytate-P
utilization, and the possibility of eliminating the need for
supplemental inorganic P, were not studied. Furthermore,
weanling pigs receiving the highest phytase activity (750
PU/g) did not sustain plasma P (inorganic) concentrations
within the normal range suggested by Ullrey et al. (1967).
By using orthogonal polynomials developed from our previous
study (Lei et al., 1992a), we predicted that the phytase
activity in a corn-soybean meal diet required to maintain
normal plasma P concentrations for weanling pigs would be
1200 PU/g. Therefore, higher levels of supplemental dietary
phytase activity, arrayed around this predicted value, were
used in two subsequent experiments. The objectives of these
studies were 1) to determine the breakpoint relating
supplemental dietary phytase activity to maximum
improvements in phytate-P utilization, and 2) to determine

the inorganic P equivalent of optimal phytase activity.

60

Materials and Methods

Phytase and Diets. The microbial phytase (A. niger,
Alko Ltd., Rajamaki, Finland) and the process of phytase

incorporation into diets were the same as described
previously (Lei et al., 1992a). The basal diet (BD) is shown
in Table 2.1 and was the same fortified corn-soybean meal
diet without supplemental inorganic P as used in Exp. 1.2 in
our previous study (Lei et al., 1992a). Mono-dibasic calcium
phosphate (MDCaP) was added to the basal diet as a positive
control. The analyzed concentrations of P, Ca, Mg, Cu, Fe,
and Zn in the experimental diets are presented in Table 2.2.
Animals and Treatments. All pigs used in the two
experiments were 4-wk old weanling crossbreds (Landrace-
Yorkshire-Hampshire). In Exp. 2.1, fifty pigs (7.61 r .56 kg
BW) were split equally into heavy and light blocks based on
body weight. Within each block, 25 pigs were allotted into
five pens of five pigs each. The BD was supplemented with
microbial phytase at 750, 1,050, 1,250, or 1,350 PU/g (one
PU is defined as the amount of enzyme that liberates 1 nmol
of inorganic P from sodium phytate per minute at Ph 5 and 37
°C) or with .21% inorganic P (MDCaP) as the positive
control. Selected dietary levels of phytase activity were

based on the predicted optimal activity (1,200 PU/g; Lei et

61

62

Table 2.1. Composition and nutritive values of the basal
diet and basal diet supplemented with mono-dibasic calcium
phosphate (MDCaP)a

 

Item Basal + MDCaP

Ingredient -------- g/kg --------
Corn (ground, shelled) 777.4 769.4
Soybean meal (44% CP) 200.0 200.0
MDCaP - 10.0
Calcium carbonate (38% Ca) 10.0 7.0
L-Lysine HCl 2.6 2.6
Salt (NaCl) 3.5 3.5
Vitamin-trace mineral premixb 3.0 3.0
Vitamin E-Se premixc 3.0 3.0
Antibiotic premixd .5 .5

Calculated nutritive values (as fed)
ME, MJ/kg 13.8 13.7
CP, g/kg 155.0 153.4
Lysine, g/kg 10.2 '10.2
Ca, g/kg 4.6 5.6
P, g/kg 3.3 5.4

a MDCaP contains not less than 21% P and 15% Ca and no more
than 18% Ca.

b Supplied the following amounts per kilogram diet: vitamin
A, 1,980 IU; vitamin D3,.396 IU; menadione, 3.3 mg;
riboflavin, 2 mg; niac1n, 11 mg; d-pantothenic acid, 8 mg;
choline, 66 mg; vitamin Bar 12 pg; Zn, 45 mg; Fe, 35 mg;
Mn, 20 mg; Cu, 6 mg; I, .12 mg.

c Supplied 10 IU of vitamin E and .2 mg of Se per kilogram
diet.

d Supplied 55 mg of chlortetracycline per kilogram diet.

63

Table 2.2. Analyzed dietary concentrations of P, Ca, and
other elementsa

Diet P Ca Mg Zn Cu Fe
"""" g/kg """‘ """‘ mg/kg '“‘“""

Experiment 1

Basal 2.9 6.5 1.5 61 12 178
+ 750 PU 3.4 7.3 1.6 69 14 205
+ 1050 PU 3.3 6.3 1.5 58 12 163
+ 1250 PU 3.1 6.5 1.6 62 13 184
+ 1350 PU 3.2 6.6 1.6 78 13 188
+ MDCaP 6.2 6.7 1.6 65 11 183

Experiment 2

Basal 3.3 5.8 73 18
+ Phytase 3.2 5.6 73 13
+ MDCaP 5.3 5.3 72 15

 

aAs fedibasis.

64
al., 1992a). Housing, management, P-depletion procedures,
and the experimental periods were the same as in the
previous study (Lei et al., 1992a).

In Exp. 2.2, twelve pigs (6.39 i . 74 kg BW) were
allotted equally into three groups receiving the BD, the BD
plus the optimal phytase activity (1,200 PU/g) determined in
Exp. 2.1 (+ Phytase), or the BD plus .21% inorganic P (+
MDCaP). Pigs were housed in individual stainless steel
metabolism cages and fed the low-P, BD (Table 2.1) for 2 wk
to adjust P reserves before the actual experimental period.
Pigs were then fed their designated diets for 2 wk.

Sample Collection spg Measurements. In Exp. 2.1,
individual pig weights and pen feed consumption were
measured weekly. Blood samples were taken weekly from three
pigs per pen for assay of plasma P, Ca , Mg, Cu, Fe, and Zn
concentrations, and plasma alkaline phosphatase (AP)
activity. In Exp. 2.2, P and Ca balance trials were
conducted as previously described (Ilori et al., 1984; Lei
et al., 1992a). Total collections of feces and urine from
individual pigs were made during the last 4 d of the trial.
Blood samples were taken from each pig initially (Wk 0), at
the end of the first wk (Wk 1), and at the end of the trial
(Wk 2) for assay of plasma P, Ca, and Zn concentrations, and
plasma AP activity. Body weights also were recorded at each
bleeding.

Assays. Concentrations of P and Ca in feed, feces,

65
urine, and plasma and activity of plasma AP were determined
by the methods previously outlined (Lei et al., 1992a).
Concentrations of other elements (Mg, Cu, Fe, and Zn) in
plasma and feed were determined by flame atomic absorption
spectrophotometry (Instrumentation Laboratory, Inc., Model
IL 951). Plasma AP activity was determined on the same day
that blood samples were drawn by the method outlined by
Sigma Chemical (1987).

Statistics. Results of Exp. 2.1 were analyzed as a
randomized complete block design with 5 treatments (4
phytase levels plus inorganic P as positive control) in 2
blocks (heavy and light BW) with repeated measurements (4
wk). Pen was considered the experimental unit. Regression
equations between dietary phytase activity and various
response measures were developed by the GLM procedure of SAS
(1988). Breakpoints of dietary phytase activity for
different measures and their inorganic P equivalents were
determined as outlined by Gill (1978). Results of P and Ca
balance in Exp. 2.2 were analyzed as a randomized complete
block design with 3 treatments. Plasma measures in Exp. 2.2
were analyzed as the same model with repeated measurements
(2 wk). Relationships between plasma and urinary measures
were also determined by the GLM procedure of SAS (1988). The
Bonferroni p-test was used for treatment mean comparisons.
The significance level was set at P < .05, unless indicated

otherwise.

Results

Experiment 2.1

Daily Gain, Daily Feed Intake and Gain/Feed. Effects of
dietary phytase activity on ADG, ADFI, and gain/feed are
presented in Table 2.3. Average daily gains of pigs
receiving MDCaP were greater (P < .05) than those of pigs
receiving the three lowest levels of phytase activity at wk
3 and greater (P g .05) than those of pigs receiving the
lowest and highest levels of dietary phytase activity at wk
4. Among the four groups of pigs receiving graded levels of
phytase, daily gains tended to increase with dietary phytase
activity at wk 3, but there was no linear relationship (2 >
.05). Daily gains of pigs at wk 4 appeared to respond to
dietary phytase activity quadratically (second degree
polynomials, g < .06, Table 2.6). Overall, pigs receiving
750 PU/g feed had the lowest and pigs receiving MDCaP had
the highest ADG. The other three groups of pigs receiving
phytase had very similar ADG. Pigs receiving 1250 PU/g feed
had ADG closest to those of pigs receiving MDCaP.

Significant differences in ADFI between different
treatment groups of pigs were observed only at wk 4. Pigs
receiving 750 PU/g feed had lower intakes (P < .05) than
pigs receiving 1250 PU/g feed or MDCaP.

Pigs receiving MDCaP tended to have a higher gain/feed

66

67

Table 2.3. Daily gain, feed intake, and feed efficiency of
pigs receiving graded levels of supplemental microbial
phytase activity or supplemental mono-dibasic calcium
phosphate (MDCaP) in the diet in experiment 2.1

Time ------ Phytase, PU/g of diet --------
750 1050 1250 1350 MDCaP

 

------------- Daily gain, g -------------—

Wk 1 172 203 226 161 221
Wk 2 242 288 247 212 320
Wk 3 285x 307x 320x 381"' 453V
Wk 4 395x 557yz 603yz 547y 660z
Overalla 273 339 349 325 414
(SEDb = 34.7, df of error = 18)
------------- Feed intake, g/d --------------
Wk 1 339 325 376 315 373
Wk 2 594 597 617 550 610
Wk 3 760 696 784 773 909
Wk 4 994x 1115"y 1268y 1138"y 1243Y
Overall 672 683 761 694 784
(SED = 70.7, df of error = 11)
------------ Gain/feed, g/kg ----------------
Wk 1 520 619 599 511 590
Wk 2 413 477 396 385 523
Wk 3 410 452 409 498 499
Wk 4 401 498 476 483 533
Overall 419 496 458 471 528

(SED = 56.0, df of error = 14)

 

a Comparison of overall means may not be used because of
interaction of treatment by time on ADG (P < .0001) and ADFI
(g < .06).

b Standard error of differences between any two treatment
means at a given wk.

“’“z Means within a row lacking a common superscript letter
differ (2 < .05).

68

than other groups of pigs, but there was no significant
difference between any two treatment groups. However, the
relationship between gain/feed and dietary phytase activity
at wk 1 and 4 fit two second degree polynomials (Table 2.6).

glsspa Inorganic P and Ca Concentrations and Alkaline
Phosphatase Activity. Effects of dietary phytase activity on
plasma P and Ca concentrations and AP activity are presented
in Table 2.4. Plasma P concentrations increased linearly
with dietary phytase activity at all weeks of the study. The
responses could be represented by four orthogonal
polynomials (Table 2.6). During the last 2 wk, pigs
receiving 1250 or 1350 PU/g feed had plasma P concentrations
near or in the proposed normal range (Ullrey et al., 1967),
but the values were only 70% of those of the pigs fed
supplemental MDCaP.

In contrast, plasma Ca concentrations of pigs decreased
linearly with increasing dietary phytase activity. Likewise,
there were four polynomials that represented these responses
(Table 2.6). However, the response of plasma Ca to dietary
phytase activity at wk 3 could be fit better by a quadratic
regression (Table 2.6). This may have resulted from the
random oscillation of a few observations, which often
happens in relatively small samples. Pigs receiving
supplemental MDCaP had lower (P < .05) plasma Ca

concentrations than pigs receiving 750 PU/g feed.

69

Table 2.4. Plasma inorganic P and Ca concentrations, and
alkaline phosphatase activity of pigs receiving graded
levels of supplemental microbial phytase activity or
supplemental mono-dibasic calcium phosphate (MDCaP) in the
diet in experiment 2.1

Time ------ Phytase, PU/g of diet --------
750 1050 1250 1350 MDCaP

 

---------- Plasma inorganic P, mg/dL ----------

Wk 0 6.2 6.4 5.5 5.4 5.8
Wk 1 5.0x 5.2x 5.4" 6.0" 8.6y
Wk 2 4.6" 5.9xy 6.0" 6.7V 9.51
Wk 3 4.8x 6.0"y 6.3y 6.5y 10.3z
Wk 4 4.5x 5.9" 6.7V 7.1V 10.2z
(SED°==.48, df of error = 16)
------------- Plasma Ca, mg/dL ---—---------------
Wk 0 9.7 10.1 10.3 10.2 12.4
Wk 1 12.8x 11.9"y 11.1” 11.4"y 10.7y
Wk 2 12.9" 11.9“ 11.6” 11.0"y 10.6y
Wk 3 13.4x 11.0y 11.2Y 11.0y 10.6Y
Wk 4 13.5" 12.3xy 12.0” 11.7xy 10.8y
(SED = .58, df of error = 12)
------- Plasma alkaline phosphatase, Ub/dL ------
Wk 0 13.4 13.3 12.1 12.7 12.6
Wk 1 17.1 16.3 16.0 16.2 13.7
Wk 2 19.6 18.5 15.3 17.9 15.6
Wk 3 20.0x 14.4” 12.3Y 16.2"y 12.5Y
Wk 4 19.6" 16.3” 13.9"y 16.1"y 11.6y

(SED = 1.88, df of error = 16)

 

a Standard error of differences between any two treatment
means at a given wk.

b Sigma unit (Sigma Procedure No. 425, 1987).

x'V'z Means within a row lacking a common superscript letter
differ (g < .05).

70

Significant differences in plasma AP activity were
observed between treatment groups during the last 2 wk of
the study. Pigs receiving 750 PU/g feed had higher plasma AP
activity (P < .05) than pigs receiving either 1250 PU/g feed
or MDCaP. Pigs receiving 1050 and 1350 PU/g feed had similar
activities, intermediate to those of pigs in the other three
groups. A quadratic relationship between plasma AP activity
and dietary phytase activity appeared at wk 3 (Table 2.6).

Plasma Mg, CuI FeI and Zn Concentrations. There was no
consistent effect of dietary phytase activity on plasma Mg,
Fe, Cu, or Zn concentrations (Table 2.5). However, the
relationship between plasma concentrations of these elements
and dietary phytase activity could be represented by second
degree polynomials (Table 2.6).

Breakpoints of Dietary Phytase Activity. All the
coefficients and levels of significance of the above
regressions between dietary phytase activity (X, PU/g) and
various response measures (Y) are summarized in Table 2.6.
The models for linear and quadratic regressions were as
follows:

Linear: Y = a + b1X;

Quadratic: Y = a + b1X + bzxz.

Breakpoints, estimates of dietary phytase activity at which
maximum responses of designated measures are expected to

occur, were derived as X = -b1/2b2 from the quadratic

71

Table 2.5. Plasma concentrations of Mg, Cu, Fe, and Zn in
pigs receiving graded levels of supplemental microbial
phytase activity or supplemental mono-dibasic calcium
phosphate (MDCaP) in the diet in experiment 2.1

Time ------ Phytase, PU/g of diet --------
750 1050 1250 1350 MDCaP

 

---------------- Plasma Mg, mg/dL ---------------

Wk 0 2.27 2.23 2.27 2.22 2.20
Wk 1 2.01 2.04 1.96 2.01 1.96
Wk 2 1.97 1.99 1.92 1.96 1.92
Wk 3 2.07y 2.07” 1.92"y 1.85" 1.99"y
Wk 4 1.92 1.87 1.83 1.90 1.89
(SED°==.071, df of error = 20)
--------------- Plasma Cu, ug/dL -------------
Wk 0 201 207 201 209 194
Wk 1 159 148 152 169 147
Wk 2 158 150 140 171 138
Wk 3 171 155 153 182 156
Wk 4 137 143 139 128 144
(SED =12.5, df of error = 14)
--------------- Plasma Fe, ug/dL ----------------
Wk 0 137 160 141 126 157
Wk 1 183 228 211 205 197
Wk 2 193 217 186 156 170
Wk 3 250 184 181 176 204
Wk 4 175 195 179 175 175
(SED = 31.8, df of error = 20)
-------------- Plasma Zn, ug/dL -----------------
Wk 0 76 95 90 89 90
Wk 1 75 82 75 74 80
Wk 2 112 106 100 103 98
Wk 3 79 74 64 80 81
Wk 4 106V 106V 106V 107V 83x

(SED = 8.8, df of error = 17)

 

a Standard error of differences between any two treatment
means at a given wk.

X'Y Means within a row lacking a common superscript letter
differ (P < .05 for Mg and g < .15 for Zn).

72
regressions, and are shown in Table 2.7. Four major
measures, ADG, gain/feed, plasma AP activity, and plasma Ca
concentrations appeared to maximize at similar dietary
phytase activities (approximately 1,200 PU/g). The estimated
maximum responses of these measures were equal to or greater
than 90% of those of the pigs fed supplemental MDCaP.
Indeed, there was no significant difference between these
two categories (Table 2.7). Breakpoints of dietary phytase
activity for plasma Mg, Cu, and Fe concentrations deviated
slightly from those for the four major measures discussed

above.

Experiment 2.2

Balance of P and Ca. Results of P and Ca balance are
presented in Table 2.8. With similar daily P intake, pigs
fed phytase retained 60% more P and excreted 55% less P in
feces than pigs fed the BD (2 < .05). Daily urinary P loss
in pigs on both diets was negligible. Thus, 99% of absorbed
P was retained in both groups of pigs. The apparent
digestibility of dietary P and the percentage of P ingested
that was retained by pigs fed phytase were 40 and 41
percentage units higher (P < .05) than those by pigs fed the
BD. On the other hand, daily P intakes of pigs fed
supplemental MDCaP were 44% higher (2 < .05) than those of

pigs fed phytase.

73

Table 2.6. Regression coefficients of different measures
with dietary phytase activity in experiment 2.1a

 

Measureb ‘Week a b1 b2 R2 P <
Quadratic
ADG 4 -862 2.45 -.001 .68 .06
Gain/feed 1 -720 2.56 -.00122 .50 .17
4 -263 1.31 -.00057 .71 .05
Plasma AP 3 74 -.11 .000047 .60 .10
Plasma Ca 3 28 -.029 .000012 .75 .03
Plasma Mg 3 1.2 .0021 -.0000012 .71 .04
Plasma Cu 3 444 -.57 .00027 .54 .15
Plasma Fe 2 -239 .93 -.00047 .67 .06
3 650 -.77 .00031 .56 .13
Linear
Plasma inorganic P
1 3.90 .0014 .38 .11
2 2.30 .0032 .79 .003
3 2.77 .0028 .41 .08
4 1.25 .0044 .90 .0004
Plasma Ca 1 14.66 -.0026 .80 .003
2 15.05 -.0029 .58 .03
3 15.87 -.0038 .59 .03
4 15.70 -.0030 .66 .01

 

a The model for linear regression is Y = Na.+-t>X and for
quadratic regression is Y = a + b1X + b X, where Y= response
measure, and X= dietary phytase activity (PU/g ).

Unit is, g for ADG, g/kg for gain/feed, U/dL for plasma
AP, mg/dL for plasma inorganic P, Ca, and Mg, and ug/dL for
plasma Cu and Fe.

74

Table 2.7. Breakpoints of dietary phytase activity for
different response measures and the comparison of these
maximum responses with those of the control pigs receiving
supplemental mono-dibasic calcium phosphate in the diet in
experiment 2.1

 

Measure Wk Break Maximum Control SEDb df P<
Pointa Response Response

ADG, g/d

4 1183 587 660 42 7 .12
Gain/feed, g/kg

1 1052 629 590 37 5 .34

4 1156 496 533 37 5 .36
Plasma AP, U/dL

3 1136 13.2 12.5 1.9 6 .72
Plasma Ca, mg/dL

3 1200 10.9 10.6 .47 7 .53
Plasma Mg, mg/dL

3 884 2.1 2.0 .06 9 .13
Plasma Cu, ug/dL

3 1036 150 156 10 7 .56
Plasma Fe, ug/dL

2 985 219 170 21 10 .04

3 1240 176 204 21 10 .21

 

3 Dietary phytase activity, PU/g of diet.
b Standard error of differences between the estimated and
control means.

75

Table 2.8. Balance of P and Ca in pigs fed the basal diet
supplemented with or without the optimal dose of microbial
phytase or mono-dibasic calcium phosphate (MDCaP) in
experiment 2.2

 

 

Item Basal + Phytase + MDCaP SEDa P <b
----------- Phosphorus, mg/d -------—---
Intake 1,092x 1,126x 1,618Y 157 .01
Fecal 792V 359x 763V 109 .006
Urinary 3x 5x 39’ 10 .05
Absorbed 300x 767’ 855y 109 .001
% of intake 28x 682 53y 5 .0001
Retained 297x 762V 816V 114 .002
% Of intake 27x 682 50y 6 .0001
% of absorbed 99 99 95 2 .07
--------------- Calcium, mg/d -----------
Intake 1,920 1,971 1,557 211 .16
Fecal 1,041Y 746“' 716x 111 .13
Urinary 370y 212“’ 63x 87 .10
Absorbed 879”' 1,225y 841x 130 .03
% Of intake 46 62 54 6 .08
Retained 509x 1,013Y 778“’ 132 .01
% of intake 27x 51’ 50y 8 .02
% of absorbed 58x 83”’ 93V 11 .04

 

a Standard error of differences between any two treatment

means.

b Significance of main effects.

x'Y'z Means within a row lacking a common superscript letter
differ (g < .05).

 

76
However, pigs supplemented with MDCaP retained only 7% more
(2 > .05) P daily because their daily fecal and urinary P
excretion was 2.1 and 7.8 times higher (P < .05),
respectively, than that of pigs fed phytase. Consequently,
the apparent digestibility of dietary P and percentage of
ingested P that was retained by pigs supplemented with MDCaP
was 15 and 18 percentage units lower (g < .05),
respectively, than that of pigs fed phytase. Based on the
differences in daily P intake and retention among these
three groups of pigs, 1,000 PU supported retention of 1.1 mg
P from the BD which was effectively equivalent to .91 mg
inorganic P as MDCaP. In addition, pigs fed MDCaP absorbed
the supplemental inorganic P almost completely because these
pigs excreted basically the same amount of P in their feces
as those pigs fed the BD.

Daily Ca intake of pigs fed the BD or BD plus phytase
was essentially the same, but daily Ca retained by the
former was only half that of the latter (2 < .05). Compared
with pigs fed phytase, pigs fed supplemental MDCaP had
slightly lower daily Ca intake. However, these two groups of
pigs retained the same proportion of ingested Ca and shared
a similar daily fecal Ca excretion. Pigs fed supplemental
MDCaP excreted less Ca in their urine and, thus, utilized

absorbed Ca somewhat better than pigs fed phytase.

77

Plasma Inorganic PI Ca, and Zn Concentrationsll and
Alkaline Phosphatase Activity. Effects of supplemental
dietary phytase and MDCaP on plasma P, Ca, and Zn
concentrations, and AP activity are presented in Table 2.9.
Initial values of these four measures among the three
treatment groups were essentially the same. However, plasma
P concentrations were different (2 < .05) during the 2 wk of
study between any two treatment groups. Pigs fed the BD
failed to sustain their initial plasma P concentrations and
had these values decreased more than 50% at the end of wk 2.
In contrast, pigs fed phytase had an increase of 1 mg/dL
above their initial plasma P concentrations, and pigs
supplemented with MDCaP had an even greater (2_< .05)
increase of plasma P concentrations during the same period.
Plasma Ca concentrations of pigs fed the BD were higher (g_<
.05) than those of pigs in the other two groups. Plasma Zn
concentrations of pigs fed phytase were higher (2_< .05) at
wk 2 than those of pigs fed MDCaP. Plasma AP activities of
pigs fed phytase and MDCaP tended to be lower (£_> .05) than

those of pigs fed the BD.

Relationship between Urinary P and Ca Excretion and

 

Plasma Inorganic P and Ca Concentration. There were moderate

correlations (; = .5 to .7) between urinary P and Ca
excretion and plasma P and Ca concentrations. Daily urinary

P excretion (Y, mg/d) was positively and negatively related

78

Table 2.9. Plasma inorganic P, Ca, and Zn concentrations and
alkaline phosphatase activity of pigs fed the basal diet
supplemented with or without the optimal dose of microbial
phytase or mono-dibasic calcium phosphate (MDCaP) in
experiment 2.2

 

Time Basal + Phytase + MDCaP
------- Plasma inorganic P, mg/dL --------
Wk 0 5.12 5.13 4.54
Wk 1 4.19x 5.49y 6.78z
Wk 2 2.54x 6.00Y 7.05z
(SEDb== .35, df of error = 8)
------------- Plasma Ca, mg/dL ---------—-
Wk 0 9.93 9.87 9.41
Wk 1 10.77y 10.07xy 8.89x
Wk 2 11.29Y 9.32x 8.56x
(SED = .44, df of error = 10)
---------- Plasma Zn, pg/dL ----------
Wk 0 64 66 63
Wk 1 89 73 76
wk 2 59xy 76V 51"
(SED = 7.3, df of error = 10)
------ Plasma alkaline phosphatase, Ub/dL ------
Wk 0 14.88 12.77 11.43
Wk 1 19.53 14.41 12.75
Wk 2 15.98 11.08 12.90

(SED = 2.81, df of error = 10)

 

a Standard error of differences between any two treatment
means at a given wk.

b Sigma unit (Sigma Procedure No. 425, 1987).

“1": Means within a row lacking a common superscript letter

differ (g < .05).

79
to plasma P and Ca concentrations (X, mg/dL), respectively:

Y = ~51 + 12.1 x (x

plasma P at wk 1, g < .06, p = .55);

Y = -19 + 6.6 X (X = plasma P at wk 2, g < .07, p = .55);
Y = 156 - 14.1 X (X = plasma Ca at wk 1, g < .05, p = .58).
In contrast, daily urinary Ca excretion (Y, mg/d) was
negatively and positively related to plasma P and Ca
concentrations (X, mg/dL), respectively:
Y = 888 - 123 X (X = plasma P at wk 1, P < .01, p = .69);
Y = 492 - 53 X (X = plasma P at wk 2, P < .07, p = .54);
Y = 109 + 52 X (X = plasma Ca at wk 1, P < .06, p = .56).

In addition, a nonsignificant negative correlation ( p = -
.4, g > .2) was found between urinary P and Ca excretion.

Daily Gain. Overall ADG during the 2 wk study was 130,
160, and 166 g for pigs fed the BD, BD plus phytase, and BD
plus MDCaP, respectively. There was no significant effect of
treatments on ADG. However, daily gain of pigs was measured
only to indicate that pigs were in good health and were

gaining weight in the experiment.

Discussion

Two questions arose after our earlier demonstration of
a significant linear effect of dietary phytase activity (up
to 750 PU/g) on ADG, ADFI, and plasma P concentrations in
young pigs (Lei et al., 1992a). The first question was how

dietary phytase activity beyond 750 PU/g would affect these

80

response measures, and how much activity would be needed to
produce maximum responses. The second was whether or not
supplements of the determined optimal dietary phytase
activity could obviate the need for inorganic P supplements
for weanling pigs fed corn-soybean meal diets. The results
of Exp. 2.1 showed no further improvement (g_> .05) in ADG,
ADFI, gain/feed, or plasma AP activity when dietary phytase
activity reached 1,050 PU/g. Quadratic relationships between
dietary phytase activity and these measures were found and
consistently revealed the maximum breakpoint to be
approximately 1,200 PU/g. However, breakpoints for dietary
phytase activity relating to plasma Mg, Cu, and Fe
concentrations were lower and inconsistent.

With all the measures taken, only pigs receiving 750
PU/g were consistently inferior to those pigs fed MDCaP.
Differences in these measures between the other three groups
of pigs receiving higher levels of phytase activity and
MDCaP were not significant in most cases. The equivalency of
estimated maximum responses of ADG, gain/feed, plasma AP
activity, and plasma Ca concentrations at the respective
breakpoints of dietary phytase activity to that of pigs fed
MDCaP were at least 90%. Indeed, plasma P concentrations in
pigs receiving 1,250 or 1,350 PU/g were only 70% of those of
pigs supplemented with MDCaP in Exp. 2.1. Significant
differences between pigs receiving 1,200 PU/g and MDCaP were

also observed in Exp. 2.2. However, the higher plasma P

81

concentrations in pigs supplemented with MDCaP may not be
particularly crucial because the balance data of Exp. 2.2
indicated that pigs fed MDCaP, ingesting 44% more P daily
than pigs fed 1,200 PU/g, retained no more than 7% of
additional P. Urinary P excretion, in addition to fecal P
excretion, was much greater in these pigs and was positively
correlated to the higher plasma P concentrations.
Differences in plasma P concentrations may be offset by the
differences in urinary P excretion in these two groups of
pigs. Alternatively, the combined improvement in absorption
and retention of dietary P in pigs receiving 1,200 PU/g as
compared to pigs receiving inorganic P supplementation may
minimize the possible adverse effect of low dietary P
intake, and hopefully of the lower plasma P concentration as
well. Moreover, plasma P concentrations in pigs receiving
1,250 or 1,350 PU/g in Exp. 2.1 were almost within the
normal range (Ullrey et al., 1967). Therefore, we may
conclude that supplements of dietary phytase activity of
1,200 PU/g could maximize phytate-P utilization by weanling
pigs and, thus, nearly, if not completely, eliminate the
need for inorganic P supplements in corn-soybean meal diets
for weanling pigs.

Cromwell (1991) suggested that supplemental dietary
phytase at 1,000 PU/g might almost eliminate the need for
inorganic P supplements for finishing pigs. If his results

together with that we obtained in this study were applied in

 

 

82

commercial swine production, a large portion of the
inorganic P supplements currently used would be saved. Of
equal or greater importance is the 50% reduction of P
excreted in swine manure, based on the decreases in fecal
and urinary P excretion in pigs fed supplemental phytase as
compared to those supplemented with MDCaP. However, this
depends on the assumption that pigs can sustain low P intake
continuously from weaning to finishing. In addition, pigs
fed 1,200 PU/g in Exp. 2.2 excreted a larger amount of Ca
via urine than pigs fed MDCaP. It is uncertain whether this
hypercalciuria was a sign of relative P deficiency
(Pointillart et al., 1987) or simply a function of the
higher Ca intake. Pigs receiving dietary phytase had
relatively higher ADFI than pigs fed MDCaP and thus ingested
more Ca. This may require a lower dietary Ca level to
balance the low total P concentration of the corn-soybean
meal diet in which inorganic P supplements are replaced by
dietary phytase. Increased dietary Ca concentration has also
been suggested to affect phytate-P utilization adversely
(Mohammed et al., 1991). Therefore, future studies should
examine both concentrations and ratios of dietary phytase,
P, Ca and.other nutrients related to P metabolism, such as
vitamin D.

The inorganic P equivalent of dietary phytase activity
found in this study was lower than that previously reported.

Leunissen and Young (1992) supplemented the diets for

83

weanling pigs with the same sources of phytase as used in
this study at 500 to 1,000 PU/g. The responses of all
criteria measured by them indicated that 500 PU increased
the availability of P equivalent to that of adding .17% of
inorganic P from calcium phosphate to diets. In a broiler
study, Schéner et al.(1991) reported that 700 PU were
equivalent to 1.0 mg P as monocalcium phosphate. The
ingredients and the P concentrations of the BD used in these
two studies were greatly different from those of the BD that
we used in this study. These may have affected the
efficiency of supplemental phytase in releasing phytate-P
from the diets. Moreover, the efficiency of phytase may be
different in pigs than in broilers. However, our estimate of
equivalency agrees with the calculations of Cromwell (1991).

The results of this study were comparable to those of
the study reported previously (Lei et al., 1992a). However,
plasma P concentrations, ADG, and ADFI were lower in the
current study. When plasma P concentrations were regressed
against dietary phytase activity, slopes at the same week
were similar, but intercepts (g) were lower in the current
study. Thus lower plasma P concentrations were found in pigs
receiving 1,250 PU/g than previously predicted (Lei et al.,
1992a). Moreover, a significant effect of dietary phytase on
plasma Ca concentration was shown only in the current study.
Three factors may have contributed to these differences: 1)

initial BW of pigs in current study was lighter; 2) dietary

84
P and Ca concentrations were not exactly the same in these
two studies; and 3) the current study was conducted in the
winter whereas the other was conducted in the summer.
Likewise, differences in plasma P concentrations of pigs
receiving the same treatment in Exp. 2.1 and 2.2 of this
study may also be attributed to the differences in initial
BW, dietary P and Ca concentrations, and feeding regime. In
addition, we found that plasma P concentration appeared to
be the most sensitive and convenient measure of the effect
of dietary phytase on phytate-P utilization in our previous
study (Lei et al., 1992a). However, in this study plasma P
concentration kept increasing linearly with dietary phytase
activity beyond the point at which other major measures were
maximized.

Dietary Ca utilization was also improved by dietary
phytase supplements, as shown previously (Nasi, 1990, Lei et
al., 1992a). Other elements such as Mg, Cu, Fe, and Zn are
also bound with phytate in corn and soybean meal and
bioavailabilities are greatly limited (Reddy et al., 1982).
Therefore, utilization of these elements might be improved
by supplemental dietary phytase as well (Nasi, 1990).
However, no such effects of dietary phytase were shown
consistently in this study. The biologically available
concentrations of these elements provided by the regular
mineral premix may have been too high for phytase to show

any effect. Special control of dietary levels of these

85
elements may be necessary to demonstrate the effect of
dietary phytase. Measures other than plasma concentrations

of these elements also may be useful.

Implications

Results of this study indicated that inorganic P
supplementation of corn-soybean meal diets for weanling pigs
could be almost completely eliminated by adding 1,200 units
of A. niger phytase activity per gram diet. Pigs receiving
this level of dietary phytase activity make near maximum
responses in daily gains, gain/feed, plasma alkaline
phosphatase activity, and plasma Ca concentrations. These
responses were approximately 90% of those of pigs fed the
regular diet supplemented with inorganic P. These pigs also
maintained plasma P concentrations in the normal range and
utilized dietary P and Ca quite efficiently. Accordingly,
inorganic P resources could be saved and P in swine manure

reduced by at least 50%.

EXPERIMENTAL SERIES III
SIMULTANEOUS IMPROVEMENTS IN PHYTATE
PHOSPHORUS AND ZINC BIOAVAILABILITY
BY SUPPLEMENTAL DIETARY PHYTASE

(Submitted to J. Nutr.)

ABSTRACT

Two experiments were conducted to determine the effects of
supplemental microbial phytase on utilization of dietary
zinc and phytate phosphorus in weanling pigs. Experiment 3.1
was a 2 X 3 factorial arrangement of treatments with 24 pigs
for 4 wk. Two levels of phytase (A. niger), 0 or 1350
units/g, and three levels of zinc, 0, 30, or 60 mg/kg as
zinc sulfate were added to a corn-soybean meal basal diet.
Weekly measures included growth performance, plasma alkaline
phosphatase activity, and plasma mineral concentrations. In
experiment 3.2, mineral balances were determined in 12 pigs
fed the basal diet or the diet with added zinc (30 mg/kg) or
phytase (1350 units/g). The results indicated that either
supplemental phytase or zinc increased plasma alkaline
phosphatase activity and plasma zinc concentrations, but
only in the absence of the other. Supplemental phytase
decreased plasma alkaline phosphatase activity in pigs
supplemented with zinc. Supplemental phytase also
significantly enhanced weight gain, feed intake, gain/feed,
plasma concentrations of inorganic phosphorus and iron, and
retention of phosphorus and calcium. Neither supplemental

phytase nor zinc affected plasma concentrations of copper

86

87
and magnesium or zinc retention. Supplementing corn-soybean
meal diets with microbial phytase at 1350 units/g feed
appears to improve bioavailability of zinc as well as of
phytate phosphorus in weanling pigs.
INDEXING KEY WORDS:
. phytase . phytate . pig

. phosphorus . zinc
Introduction

Over 50% of phosphorus in corn, soybean meal, and other
plant seeds is in the form of phytate (pyp-inositol
phosphates) that is poorly available to pigs and other
simple-stomached animals (Reddy et al. 1982). Consequently,
provision of an inorganic phosphorus supplement in pig diets
composed mainly of these ingredients is routine (NRC 1988).
This supplement not only increases diet cost but may result
in accumulation of excess phosphorus in cropland to which
swine manure is applied (Cromwell 1991). Of significance in
both human and animal nutrition is the formation of
insoluble complexes of phytate with zinc at certain
concentrations of calcium in the digestive tract, thereby
greatly inhibiting zinc absorption (O'Dell and Savage 1960,
Simpson and Wise 1990). Thus, typical corn-soybean meal
diets for swine do not provide sufficient available zinc to

meet the requirement (NRC 1988) even though these diets

88

contain a level of zinc that would be adequate for pigs
consuming a casein—glucose diet (Shanklin et al. 1968).
Indeed, removal of phytate from infant soy formulas
increased zinc absorption from 16% to 47% in suckling rats
and from 27% to 45% in monkeys (Lonnerdal et al. 1988).

Phytases, myp-inositol hexaphosphate phosphohydrolases
(E.C. 3.1.3.8), catalyze the stepwise removal of inorganic
orthophosphate from phytate and produce five classes of
intermediate products, myp-inositol pentakis-, tetrakis-,
tris-, bis-, and monophosphates (Gibson and Ullah, 1990).
Supplements of microbial phytase (Jongbloed et al. 1992, Lei
et al. 1992a, Nasi 1990, Nelson et al. 1971, Simons et al.
1990) or cereal phytase (Pointillart et al. 1987) in diets
for pigs and poultry effectively improves phytate phosphorus
utilization. In our earlier study (Lei et al. 1992b), we
demonstrated that supplementing corn-soybean meal diets with
A. niger phytase at 1200 phytase units (PU)/g of feed
appeared to maximize utilization of phytate phosphorus and
may obviate almost completely the need for inorganic
phosphorus supplementation in weanling pigs. Since
bioavailability of zinc in foods of plant origin is a
function of phytate concentration (Bobilya et al. 1991,
Ferguson et al. 1989, Lonnerdal et al. 1988), we
hypothesized that enhanced dietary phytate phosphorus
utilization by supplemental microbial phytase might also

increase the bioavailability of zinc. Two experiments were

89
conducted with weanling pigs to determine whether phytase
supplements would produce simultaneous improvements in the
bioavailability of zinc as well as phytate phosphorus in a

corn-soybean meal diet.

Materials and Methods

Phytase. Zinc. and Disps. The microbial phytase used in
this study was produced by a genetically modified strain of
A. niger (var. ficuum, 3-phytase). The enzyme product was
kindly provided by Alko Ltd., Rajamaki, Finland and phytase
activity was approximately 500,000 PU/g. Actual phytase
activity was confirmed as previously described (Lei et al.
1992a) before the product was mixed with other ingredients
in the preparation of the complete diet. One PU is defined
as the amount of enzyme that liberates 1 nmol of inorganic P
from sodium phytate per minute at pH 5 and 37 °C. The basal
diet (BD) was a fortified corn—soybean meal diet without
supplemental inorganic phosphorus or zinc (Table 3.1). The
analyzed concentrations of different minerals in the
experimental diets are also listed in Table 3.1. Zinc
sulfate (Znsopanho, ACS grade, Columbus Chemical
Industries, Columbus, WI) was used as the supplemental zinc

source .

90

 

 

TABLE 3.1
Composition and nutrient values of the basal diet1
Item Concentration
Ingredient gzkg
Corn (ground, shelled) 773.4
Soybean meal (44% CP) 200.0
Calcium carbonate (38% Ca) 10.0
L-Lysine HCl 2.6
Salt (NaCl) 3.5
Vitamin-trace mineral premix2 5.0
Vitamin E-Se premix3 5.0
Antibiotic premix‘ .5
Analyzed zinc concentration (as fed) mgzkg
Added
Diet Phytase Zinc Total zinc
PU/g mg/kg mg/kg
Experiment 15
1 0 0 27
2 0 30 50
3 0 60 86
4 1350 0 25
5 1350 30 47
6 1350 60 94
Experiment 26
Basal 0 0 30
+ Zinc 0 30 55
+ Phytase 1350 0 32

1 The basal diet provided all nutrients at recommended
concentrations (NRC, 1988) with the exception of phosphorus,
calcium, and zinc.
2 Vitamin-trace mineral premix (g/kg): vitamin A acetate
(30,000 IU/g), 22; vitamin D3 (3000 IU/g), 44; menadione
sodium bisulfite, 0.44; riboflavin, 0.66; niacin, 3.52;
d-pantothenic acid, 2.64; choline chloride, 36.56; vitamin
B12 (triturated), 3.96; MnO, 11.45; KIO3, 0.18; CuSanSHZO,
7.93; FeSOpflhO, 35.68; butylated hydroxytoluene, 9.91.
3 Vitamin E-Se premix: a-tocopheryl acetate (500 IU/g), 0.15
g/kg; NaZSeO3, 44 mg/kg.

Supplied 55 mg of chlortetracycline per kilogram diet.
5 The basal diet was analyzed to contain per kilogram:
phosphorus, 2.9 g, calcium, 4.6 g, magnesium, 1.6 g, zinc,
27 mg, copper, 10 mg, and iron, 108 mg.
6 The basal diet was analyzed to contain per kilogram:
phosphorus, 3.0 g, calcium, 4.7 g, zinc, 30 mg, and copper,
15 mg.

91

Animals and Treatments. All pigs used in the two
experiments were weanling crossbreds (Landrace-Yorkshire-
Hampshire). Experimental housing was maintained at 22-25 W3,
with a 12 h light:dark cycle. In experiment 3.1, twenty four
pigs (4-wk old, 7.38 i .37 kg body weight) were reared in 12
stainless steel metabolism pens (two pigs per pen) with
slotted floors. A 2 X 3 factorial arrangement of treatments
with 2 levels of supplemental phytase, 0, and 1350 PU/g, and
3 levels of supplemental zinc, 0, 30, and 60 mg/kg in the BD
was conducted for four wk. Pigs were given ad libitum access
to feed and water. All pigs were fed the low phosphorus and
zinc BD for 10 d to deplete partially the phosphorus and
zinc reserves before the formal trial was conducted.

In experiment 3.2, twelve pigs (3-wk old, 6.00 i .62 kg
body weight) were allotted equally into groups receiving the
BD, the BD plus zinc, 30 mg/kg (+ Zinc), or the BD plus
phytase activity, 1350 PU/g (+ Phytase). Pigs were housed in
individual stainless steel metabolism cages and fed the BD
for two wk to deplete the phosphorus and zinc reserves
somewhat before the actual experimental period. Pigs were
then fed their designated diets and given ad libitum access
to distilled water for 20 d.

Sample Collection spg Measurements. In experiment 3.1,
individual pig weights and pen feed consumption were
measured weekly. Blood samples of all pigs were taken weekly

from the anterior vena cava for assay of plasma inorganic

92
phosphorus, calcium, magnesium, copper, iron, and zinc
concentrations, and plasma alkaline phosphatase (AP,
E.C.3.1.3.1) activity. In experiment 3.2, phosphorus,
calcium, and zinc balance trials were conducted as
previously described (Lei et al. 1992a). Total collections
of feces and urine from individual pigs were made during the
last 4 d of the trial. Dietary intake and fecal and urinary
mineral excretion rates for the balance period were
calculated as the average per kilogram body weight per day
for each animal (Kimmel et al. 1992). Blood samples were
taken from each pig at d 0, d 10, and at d 20 for assay of
plasma phosphorus, calcium, and zinc concentrations, and
plasma AP activity. Body weights also were recorded at each
bleeding.

Assays. Concentrations of phosphorus in feed, feces,
urine, and blood plasma were determined by a colorimetric
method (Gomori 1942), and concentrations of calcium and zinc
in these samples plus other elements in plasma and feed were
determined by flame atomic absorption spectrophotometry
(Model IL 951, Instrumentation Laboratory, Inc., Wilmington,
MA). Plasma AP activity was determined on the day that blood
samples were drawn by the method outlined by Sigma Chemical
(1987).

This study was approved by the All-University Committee
on Animal Use and Care of Michigan State University.

Statistics. Data from experiment 3.1 were analyzed as a

93
split-plot model with factorial arrangement of treatments
and time repeated measurements (Gill, 1986). Pen was
considered the experimental unit. Balance of phosphorus,
calcium, and zinc in experiment 3.2 were analyzed as a
randomized complete block model with 3 treatments. Plasma
measures in experiment 3.2 were analyzed as a split-plot
model with repeated measurements (d 10 and d 20). The
Bonferroni p-test was used for conditional comparisons of
treatment means. Standard errors of mean differences,
instead of standard errors of single means, for all measures
were listed as recommended by Gill (1986). Interactions were
considered to have modest importance if g < 0.20, but
primary comparisons were declared significant only if 2 <

.05, unless indicated otherwise.

Results

Experiment 3.1

Main Effects. Statistical analyses of the main effects of
supplemental phytase and zinc and their interactions on
various measures are presented in Table 3.2. Supplemental
phytase and zinc interacted to affect plasma AP activity (P
< 0.005), plasma zinc concentrations (2 < 0.01), and daily

feed intake (2 < 0.09). In addition, supplemental phytase

94

TABLE 3.2
Significances and standard errors of mean differences of
main effects of dietary phytase and zinc, and their
interactions on various measures1

 

 

 

Significance Standard error of
P < mean difference2
Measures Phy3 Zn Phy* Zn Phy Zn Phy*Zn df“

 

Growth perforamces

Weight gain 0.0006 0.83 0.44 32 40 56 16
Feed intake 0.0001 0.58 0.09 53 64 91 24
Gain/feed 0.05 0.89 0.96 38 46 66 19

Plasma alkaline phosphatase activity
0.06 0.002 0.005 13.7 16.8 23.7 15

Plasma mineral concentrations

Phosphorus 0.0001 0.07 0.67 0.09 0.11 0.15 23
Calcium 0.05 0.21 0.87 0.12 0.14 0.20 24
Zinc 0.0005 0.0008 0.01 1.25 1.54 2.17 23
Copper 0.45 0.75 0.33 1.23 1.52 2.14 15
Iron 0.03 0.97 0.67 4.59 5.63 6.85 22
Magnesium 0.22 0.22 0.35 0.08 0.10 0.13 20

 

1 Data were analyzed as split-plot model with 2 X 3 factorial

arrangement of treatments and time repeated measurements.
Units for standard error of mean differences of various

measures are the same as described in the following tables.

3 Phytase.

‘ Calculated degrees of freedom of error.

95
affected all other measures (2 < 0.05) except plasma copper
and magnesium concentrations. However, supplemental zinc
tended to affect only plasma phosphorus concentrations (2 <
0.07) beside its interactive effects with supplemental
phytase on plasma AP activity, plasma zinc concentrations,
and feed intake. Time affected (2 < 0.01) all the response
measures taken. Strong interactions of time and phytase (P <
0.004) were observed on weight gain, feed intake, plasma AP
activity, and plasma concentrations of phosphorus and zinc.
Interactions of time and zinc were found on plasma zinc
concentrations (2 < 0.03) and plasma AP activity (g <
0.0003). Interactions of time both with phytase and zinc
appeared for gain/feed (P < 0.004), weight gain (2 < 0.1),
plasma AP activity (P < 0.08), and plasma concentrations of
zinc (g < 0.2).

Growth Performance. Effects of supplemental phytase and
zinc on daily feed intake, weight gain, and gain/feed are
presented in Table 3.3. Supplemental phytase tended to
increase daily feed intake, but the increase varied with
time and dietary zinc concentrations. In wk 4, supplemental
phytase increased (2 < 0.05) daily feed intake at all three
levels of dietary zinc. However, in wk 1 and wk 3,
supplemental phytase increased daily feed intake only in
pigs supplemented with zinc at 30 mg/kg. Phytase produced no

significant increase in feed intake in wk 2. Supplemental

Daily feed intake,

activity and zinc1

96

TABLE 3.3

weight gain,

and gain/feed of pigs
receiving different dietary levels of supplemental phytase

 

 

 

Phytase, flz/g 0 1350
Zinc, mgzkg 0 30 60 0 30 60
Feed intake, gig
'Wk 1 550 466 539 672 698 557
Wk 2 729 878 729 885 929 904
Wk 3 846 807 878 1034 1138 1023
Wk 4 927 774 968 1341 1365 1216
Overall3 763 731 778 983 1032 925
Weight gain, gig
Wk 1 343 268 273 399 453 438
Wk 2 321 414 397 524 562 456
Wk 3 416 326 412 537 544 484
Wk 4 411 369 424 667 606 589
Overall3 373 344 376 532 541 492
Gainzfeed, gzkg
Wk 1 625 575 501 588 644 786
Wk 3 491 405 469 521 477 473
Wk 4 443 475 441 499 443 486
Overall3 488 470 484 543 524 532

 

1 Data were analyzed as split-plot model with 2 X 3
factorial arrangement of treatments and time repeated
measurements. Significances and standard error of mean

differences of treatment factors are listed in Table 2.

Values are means of four pigs.
2 Phytase unit (see text for definition).
3 Comparisons of overall means should not be taken because
of interactions of treatments with time on the three

measures .

97
phytase greatly improved weight gain (2 < 0.05) in all 4 wk
of the study, independent of dietary zinc concentrations.
Supplemental phytase also improved gain/feed (g < 0.05)
during the first 2 wk of the study. In contrast,
supplemental zinc alone apparently did not affect weight
gain or gain/feed.

Plasma Alkaline Phosphatase Activitv. Effects of
supplemental phytase and zinc on plasma AP activity are
presented in Table 3.4. Pigs fed the BD had the lowest
plasma AP activity. Supplementing the BD with zinc at either
30 or 60 mg/kg increased plasma AP activity (P < 0.05) in
all 4 wk of the study, but the increase was shown only in
pigs receiving no dietary phytase. The three groups of pigs
supplemented with phytase had similar plasma AP activity.
Unlike supplemental zinc, supplemental phytase did influence
plasma AP activity at all levels of dietary zinc. Without
supplemental zinc, phytase increased (P < 0.1) plasma AP
activity in the first 3 wk. With supplemental zinc, phytase
decreased (2 < 0.05) plasma AP activity in the last 2 wk.

Plasma Concentrations of Minerals; Effects of supplemental
phytase and zinc on different element concentrations in
plasma are presented in Tables 3.4 and 3.5. Pigs fed the BD
had the lowest plasma zinc concentrations. Supplementing the
BD with either phytase or zinc increased (P < 0.05) plasma

zinc concentrations in all 4 wk of the

98

TABLE 3.4
Plasma alkaline phosphatase activity and plasma zinc,
phosphorus, and calcium concentrations in pigs receiving
different dietary levels of supplemental phytase activity
and zinc1

 

J

 

 

Phytase, fizz/g o 1350
Zinc, mgfikg 0 30 60 0 30 6o

 

Plasma alkaline phosphatase, gig.

 

Wk 0 132 164 138 147 121 149
Wk 1 99 194 185 155 157 175
Wk 2 68 201 142 127 151 154
Wk 3 66 257 190 129 148 140
Wk 4 103 286 228 119 134 143
Plasma zinc, smolzL
Wk 0 9.2 9.9 8.9 8.5 7.7 8.5
Wk 1 5.9 12.9 15.7 14.3 16.3 14.8
Wk 2 4.3 12.5 13.5 13.7 15.9 16.8
‘Wk.3 3.5 14.8 14.5 J14.5 16.6 19.7
Wk 4 3.5 10.8 13.7 13.1 16.3 13.5
Plasma phosphorus, mmoléL
Wk 0 1.7 1.6 1.6 1.5 1.7 1.5
Wk 1 1.4 1.6 1.4 2.0 2.2 1.7
Wk 2 1.0 1.1 1.1 2.0 2.0 2.1
Wk 3 1.0 1.0 0.8 1.9 2.0 2.0
Wk 4 0.8 0.7 0.7 2.0 2.1 1.7
Plasma calcium, mmolzL
Wk 0 2.9 2.8 2.8 2.7 2.7 3.0
Wk 1 3.0 2.9 3.1 2.6 3.0 2.9
Wk 2 2.9 3.0 3.1 2.8 2.7 2.8
Wk 3 2.7 2.9 2.6 2.8 2.7 2.8
Wk 4 2.4 2.6 2.7 2.3 2.3 2.7

 

(Continued on next page)

99

(Continued, TABLE 3.4)

1 Data were analyzed as split-plot model with 2 X 3
factorial arrangement of treatments and time repeated
measurements. Significances and standard error of mean
differences of treatment factors are listed in Table 2.
Values are means of four pigs.

2 Phytase unit (see text for definition).

3 Sigma unit (Sigma Procedure No. 425, 1987).

100

TABLE 3.5
Plasma iron, copper, and magnesium concentrations in pigs
receiving different dietary levels of supplemental phytase
activity and zinc1

 

 

Phytase, flz/g 0 1350
Zinc, ngKQ 0 30 60 0 30 60

 

Plasma iron, smolzL

 

Wk 0 36.4 39.8 42.1 49.6 46.1 41.3
Wk 1 42.0 35.2 36.8 40.5 46.8 38.9
Wk 2 33.4 34.6 43.9 43.9 48.2 41.6
Wk 3 28.2 37.1 32.7 51.1 37.3 43.0
Wk 4 39.3 46.3 39.6 53.8 49.5 51.6
Plasma copper, smolgL
Wk 0 22.5 21.3 21.7 22.5 21.1 23.0
Wk 1 20.8 21.3 21.3 21.9 21.2 19.2
Wk 2 20.5 20.0 19.5 25.3 19.7 23.1
Wk 3 19.5 21.3 21.1 23.4 20.3 20.9
WK 4 16.6 19.1 22.5 18.3 19.5 20.5
Plasma magnesium, mmolZL
Wk 0 1.5 1.4 1.4 . 1.4
Wk 1 1.3 . . . .5 1.3
Wk 2 1.4 1.1 . . . 1.3
Wk 3 1.5 . 1.3
Wk 4 1.2 1.0 . 1.0 . 1.2

 

1 Data were analyzed as split-plot model with 2 X 3
factorial arrangement of treatments and time repeated
measurements. Significances and standard error of mean
differences of treatment factors are listed in Table 2.
Values are means of four pigs.

2 Phytase unit (see text for definition).

101
study. However, the increases resulting from supplemental
phytase appeared only in the absence of supplemental zinc
and vice versa. Plasma zinc concentrations in the three
groups of pigs receiving phytase were similar. Higher
supplemental zinc (60 mg/kg) did not increase plasma zinc
concentrations further at either dietary phytase level. Pigs
fed phytase maintained normal plasma phosphorus
concentrations, as shown previously (Lei et al. 1992b),
through the entire study. Meanwhile, pigs receiving no
dietary phytase had plasma phosphorus concentrations that
gradually decreased to less than half of those in pigs fed
phytase (P < 0.05) by the end of the study. Dietary phytase
also increased plasma iron concentrations (2 < 0.05) in wk 3
and wk 4 and decreased plasma calcium concentrations (3 <
0.05) in wk 2. The only effect of dietary zinc alone was on
plasma phosphorus concentrations in wk 2. Pigs had higher
plasma phosphorus concentrations (2 < 0.05) when
supplemented with zinc at 30 mg/kg rather than at 60 mg/kg.
However, neither supplemental phytase nor zinc consistently

affected plasma concentrations of copper or magnesium.

Experiment 3.2
Mineral Balance. Balances of phosphorus, calcium, and zinc
in pigs fed the BD, the BD plus zinc, or the BD plus phytase

are presented in Table 3.6. With essentially the same

102

TABLE 3.6
Balance of phosphorus, calcium, and zinc in pigs fed the
basal diet or basal diet supplemented with zinc sulfate or
microbial phytase1

Item Basal + Zinc2 + Phytase3 SED” P <5

 

Phosphorus, mmollkg-d

Intake 3.29 3.33 3.42 0.31 0.91
Fecal6 2.46b 2.568 1.048 0.18 0.0001
Urinary 0.01 0.02 0.03 0.01 0.49
Retained 0.828 0.758 2.35b 0.23 0.0001
2 of intake 24.928 22.528 69.59b 4.69 0.0001
Calcium, mmolzkgod
Intake 3.86 4.22 3.98 0.38 0.65
Fecal 2.31'8 2.63b 1.258 0.29 0.002
Urinary 1.05 0.78 0.55 0.27 0.23
Retained 0.508 0.818 2.18b 0.18 0.0001
% of intake 12.958 19.198 54.778 4.67 0.0001
Zips, pmollkgod
Intake 15.178 29.138 17.418 2.32 0.0004
Fecal 20.26 31.72 22.69 5.47 0.14
Urinary 1.89 1.57 1.98 0.62 0.79
Retained - 6.98 - 4.16 - 7.26 4.86 0.79
% of intake -46.01 -14.28

-41.70 29.91 0.48

 

1 Data were analyzed as random complete block model with
three treatments. Values are means of four pigs.

2 Supplemented with 30 mg zinc as ZnS04-7HZO per kilogram
basal diet.

3 Supplemented with A. niger phytase at 1350 units/g of
basal diet.

4 Standard error of differences between any two treatment
means.

5 Significance of main effects.

6 Means within a row lacking a common superscript letter
differ (2 < .05).

103
dietary intake, pigs fed the BD plus phytase retained more
than twice as much phosphorus and calcium (2 < 0.05) daily
than the other two groups. As expected, daily fecal
excretions of phosphorus and calcium were markedly reduced
in pigs supplemented with phytase. Urinary excretions of
phosphorus were lowest in pigs fed the BD followed by pigs
supplemented with zinc and phytase. Urinary excretions of
calcium were in the opposite order. However, no significant
difference between treatments was found in urinary
excretions of these two elements. Fecal and urinary
excretions and retentions of phosphorus and calcium in pigs
fed the BD were not different from pigs fed the BD plus
zinc. All three groups of pigs were in negative zinc
balance. Due to the higher dietary zinc intake (2 < 0.05),
pigs supplemented with zinc had less negative balance and
greater fecal zinc excretions than the other two groups of
pigs. Urinary zinc excretions were similar in the three
treatment groups. In all, neither excretion via feces or
urine nor retention of zinc was significantly different
among the treatment groups.

Plasma Concentrations of Minerals and Alkaline Phosphatase
Activitv. Effects of supplemental phytase and zinc on plasma
phosphorus, calcium, and zinc concentrations, and plasma AP
activity are presented in Table 3.7. Pigs supplemented with

phytase maintained normal plasma phosphorus concentrations

104

TABLE 3.7
Plasma inorganic phosphorus, calcium, and zinc
concentrations and plasma alkaline phosphatase activity of
pigs fed the basal diet or basal diet supplemented with zinc
sulfate or microbial phytase in experiment 3.21'2

Time Basal + Zinc3 + Phytase‘

 

Plasma inorganic phosphorus, mmolZL

D 0 1.5 1.6 1.7
D 105 1.588 1.48 1.88
D 20 1.48 1.28 2.38

(SE08 = .13, df of error = 8)
Plasma calcium, mmolZL

D 0 2.5 2.4 2.4
D 10 2.8'8 2.688 2.48
D 20 2.98 2.788 2.38
(SED = .10, df of error = 9)

Plasma zinc, smolzL

D 0 9.7 9.5 10.3
D 10 10.2 11.9 10.8
D 20 8.98 11.988 12.58

(SED = 1.18, df of error = 8)
Plasma alkaline phosphatase, gflg;

D 0 145 121 135
D 10 161 177 178
D 20 127 102 101

(SED = 27.1, df of error = 10)

 

1 Data were analyzed as split-plot model with three
treatments and time repeated measurements. Values are means
of four pigs.

2 Significance of main effects of treatment on: plasma
phosphorus (P < 0.0006), plasma calcium (2 < 0.002), plasma
zinc (2 < 0.1), and plasma alkaline phosphatase (2 < 0.97).
3 Supplemented with 30 mg zinc as ZnSOpfinhO per kilogram
basal diet.

(Continued on next page)

105

(Continued, TABLE 3.7)

‘ Supplemented with A. niger phytase at 1350 units/g of
basal diet.

5 Means within a row lacking a common superscript letter
differ (2 < 0.05).

6 Standard error of differences between any two treatment
means at a given time.

7 Sigma unit (Sigma Procedure No. 425, 1987).

106

which were higher (2 < 0.05) than those in pigs fed the BD
or the BD plus zinc. Plasma calcium concentrations in pigs
receiving phytase were lower (2 < 0.05) than in pigs fed the
BD. Plasma calcium concentrations in pigs fed the BD or the
BD plus zinc were not significantly different. Plasma zinc
concentrations in pigs fed the BD were lower than those of
pigs supplemented with phytase (2 < 0.05) and zinc (P < 0.1)
at the end of the experiment. The latter two groups had
similar plasma zinc concentrations. Unlike the case in
experiment 3.1, plasma AP activity was not affected by
dietary treatments.

Weight Gain. All pigs were in good health and gaining
weight through the entire experiment. The overall daily
gains in pigs fed the BD, the BD plus zinc, and the BD plus

phytase were 125, 123, and 151 g, respectively.

Discussion

Typical corn-soybean meal diets for pigs contain
approximately 3 g of total phosphorus/kg, of which 60%, 10%,
and 6% is as myp-inositol hexakis, pentakis, and
tetrakisphosphate, respectively (Jongbloed et al. 1992, Lei
et al. 1992a and b, Simons et al. 1990). Phytase activity in
the stomach and small intestine, either from the dietary
ingredients and/or mucous cells, is negligible (Jongbloed et

a1. 1992). Substantial dietary phytate generally passes to

107
the large intestine intact, where microflora may break it
down, but phosphorus absorption is limited (Jongbloed 1987).
Therefore, dietary phytate phosphorus is mainly excreted in
the feces. As shown in previous studies (Jongbloed et al.
1992, Lei et al. 1992b, Nasi 1990, Simons et al. 1990) as
well as in this study, no more than a quarter of the total
phosphorus in these diets is retained by pigs. As expected,
pigs fed such diets develop phosphorus deficiency as
indicated by low feed intake, poor growth rate and
efficiency, hypophosphatemia, hypophosphaturia,
hypercalcemia, hypercalciuria, and elevated plasma AP
activity (Lei et al. 1992a and b, Pointillart et al. 1987).
Pigs receiving no supplemental phytase in this study showed
nearly all of these signs except elevated plasma AP
activity, which was probably limited by the low available
zinc in the BD. Supplements of inorganic phosphorus in these
corn-soybean meal diets overcome phosphorus deficiency and
support normal performance (Lei et al. 1992b). Nevertheless,
pigs supplemented with inorganic phosphorus still excrete
the same, or a greater, amount of fecal phosphorus (Lei et
al. 1992b). Thus, supplemental inorganic phosphorus does not
resolve the problem of excess phosphorus in manure
originating from the poor digestibility of phytate
phosphorus in the foresegments of the gastrointestinal tract
of pigs. Alternatively, supplementing corn-soybean meal

diets with sufficient microbial phytase activity, such as

108
1200 PU/g in our previous study (Lei et al. 1992b) and 1350
PU/g in this study, appears to release sufficient phosphorus
from phytate to meet the requirement of pigs. Of equal or
greater importance, fecal phosphorus excretion is reduced by
more than 50%. In addition, dietary calcium utilization is
also improved. Therefore, our studies, together with those
of others (Jongbloed et al. 1992, Nasi 1990, Simons et al.
1990), indicate that judicious use of microbial phytase in
swine diets may not only greatly reduce the need for
supplements of inorganic phosphorus, a nonrenewable resource
and the third largest expense in the diet, but also
alleviate excess manure phosphorus pollution which is a
severe problem facing the swine industry (Cromwell 1991).
Effects of supplemental zinc on plasma phosphorus
concentrations were observed only in wk 2 of experiment 3.1.
Basically, the positive effects of supplemental phytase on
phytate phosphorus utilization in this study appeared to be
independent of dietary zinc concentration. However, zinc
deficiency has been shown to modulate biochemical responses
to dietary phosphorus and calcium in rats (Kimmel et al.
1992).

Zinc in foods of plant origin is less bioavailable than
that of animal origin because of the adverse effect of
phytate on zinc utilization (Bobilya et al. 1991, Lonnerdal
et al. 1988). Removal or reduction of phytate in food

generally improves zinc bioavailability (Lonnerdal et al.

109
1988, Reddy et al. 1982). However, attempts to enhance zinc
utilization in vivo by supplementing the diet with microbial
phytase have not been previously reported. In this study, we
demonstrated that supplements of A. niger phytase, just as
supplements of zinc, in a corn-soybean meal BD greatly
increased plasma zinc concentrations in both trials. We
attribute this increase to the effect of supplemental
phytase on phytate degradation and to zinc release.
Supplemental phytase did not affect plasma zinc
concentrations when highly available zinc was provided in
the same diets.

Improvement of zinc utilization by phytase was also
illustrated by the responses of plasma AP activity with
different treatments. Alkaline phosphatase, a zinc
containing enzyme, plays a key role in phosphorus
metabolism. Activity of AP in plasma generally increases
with phosphorus deficiency (Boyd et al. 1983, Miller et al.
1964) and decreases with zinc deficiency (Miller et al.
1968, Shanklin et al. 1968). Pigs receiving no supplemental
phytase in experiment 1 were subjected to suboptimal
available phosphorus. Thus, elevated plasma AP activity in
these pigs would be expected. However, increased plasma AP
activity occurred only in pigs supplemented with zinc. In
contrast, plasma AP activity in pigs unsupplemented with
zinc decreased. Plasma AP activity in pigs supplemented with

phytase was normal (Miller et al. 1968) and was unaffected

110
by supplemental zinc. Therefore, supplementing microbial
phytase at 1350 PU/g appeared to release not only sufficient
phytate phosphorus but also sufficient zinc from the BD for
pigs to maintain normal plasma AP activity. In contrast,
phosphorus deprivation in pigs unsupplemented with phytase
resulted in increases in plasma AP activity when highly
available zinc (i. e. zinc sulfate) was provided. Yet, the
increase was apparently limited in pigs fed the BD in which
all zinc was from plant origin. We are uncertain why plasma
AP activity failed to respond to supplemental dietary
phytase or zinc in experiment 3.2.

Criteria for evaluating zinc bioavailability are not
well established because it is still difficult to relate
zinc deficiency signs and the biochemical roles of zinc in
the body, particularly at the molecular level (Miller et al.
1991). Baker (1991) pointed out that plasma zinc
concentration and AP activity were often misleading criteria
of zinc bioavailability. He suggested that the best measures
were probably body weight gain and bone zinc accumulation
which indeed have been shown to be reliable in recent
studies (Hunt and Johnson 1992, Wedekind et al. 1992). In
our study, supplemental phytase significantly improved
growth rate and efficiency. The improvement may have
resulted from enhanced utilizations of dietary phosphorus
and zinc by phytase. Supplemental zinc alone was not shown

to promote performance directly in this study. The

111
phosphorus deficiency in pigs unsupplemented with phytase
may have precluded the possible growth-promotion effect of
supplemental zinc. The relatively low available dietary
phosphorus and high growth rate in pigs supplemented with
phytase may essentially have prevented supplemental zinc
from further improving growth or feed intake. A linear
increase in serum zinc concentration, instead of weight
gain, was demonstrated by Miller et al. (1981) in weanling
pigs supplemented with zinc from zinc oxide or metallic zinc
dust at 25 and 50 mg/kg of feed. Responses of plasma zinc
concentration to dietary zinc intake was found to agree with
those of zinc retention, femur zinc concentration, and
weight gain in neonatal pigs fed different sources of zinc
(Bobilya et al. 1991). Earlier, serum alkaline phosphatase
activity was successfully used in our laboratory as a
criterion of zinc requirement of pigs (Miller at al. 1968,
Shanklin et al. 1968). Currently, plasma alkaline
phosphatase activity has been proposed as a useful indicator
for monitoring zinc deficiency in humans (Ishikaza et al.,
1981). Most importantly, responses of plasma AP activity and
plasma zinc concentration to supplemental phytase and zinc
in this study were very consistent. Thus, we consider that
the changes of these two measures represented the
improvement of dietary zinc bioavailability by supplemental

phytase.

Zinc balance is also used to determine zinc requirement

112
and bioavailability (Bobilya et al. 1991, Shanklin et al.
1968). Nasi (1990) demonstrated no improvement in zinc
retention associated with dietary phytase supplement in
pigs. Likewise, pigs supplemented with phytase at 1350 PU/g
in the BD in this study were in negative zinc balance as
were pigs fed only the BD. It is difficult to explain how
supplemental phytase increased plasma zinc concentrations
without enhancing dietary zinc utilization, at least
absorption. Moreover, even pigs supplemented with zinc at 30
mg/kg diet were still in negative zinc balance. In fact,
total zinc concentration in the zinc-supplemented diet (55
mg/kg) was below the requirement (80 mg/kg, NRC 1988).
However, pigs supplemented with zinc at 30 mg/kg had plasma
zinc concentrations and plasma AP activities similar to
those of pigs supplemented with zinc at 60 mg/kg. Besides
the suboptimal intake of zinc, endogenous zinc excretion
(Bobilya et al. 1991, Newland et al. 1958) may have
contributed to the negative zinc balance. Ziegler et al.
(1987) reported that half of the total fecal zinc in infancy
originated from endogenous zinc excretion. Differences in
phosphorus status between pigs supplemented with and without
phytase also may have complicated zinc balance. Tao and
Hurley (1975) found that low-calcium intakes resulted in
release of skeletal zinc during bone resorption. Likewise,
the low-phosphorus intake of pigs unsupplemented with

phytase in this study may also have initiated bone

113

resorption, or at least reduced bone formation, and thus
freed some zinc. The relative excess of zinc may have
decreased dietary zinc absorption and(or) increased
endogenous zinc excretion. In contrast, high levels of
dietary phosphorus have been shown to increase fecal zinc
excretion (Greger and Snedeker, 1980). The relatively higher
available phosphorus intake of pigs supplemented with
phytase, as compared to those that were unsupplemented, may
have increased fecal zinc excretion somwhat, and thus,
offset or minimized the expected positive effect of phytase
on zinc absorption. Overall, it is not unusual to observe a
negative balance of trace elements tested over a short
period of time (Mertz 1987). Intrinsic and(or) extrinsic
labeling of zinc in food may permit better estimates of the
true bioavailability of zinc (Fairweather-Tait et al. 1991).

Besides zinc, phytate also chelates with other cations
(Reddy, 1982). Although supplemental phytase appeared to
increase only plasma iron concentrations, appropriate
experimental designs may demonstrate improvement in
bioavailability of other mineral elements by supplemental
phytase. In conclusion, supplements of microbial phytase at
1350 PU/g diet appear to release sufficient zinc and
phosphorus from a corn-soybean meal diet for pigs to
maintain normal plasma phosphorus and zinc concentrations
and normal growth and feed utilization without supplements

of either inorganic phosphorus or zinc. Whether the released

114
zinc is sufficient to maintain normal zinc balance and bone

zinc deposition in pigs deserves further study.

Implications

Supplementing corn-soybean meal diets with sufficient
microbial phytase activity appears to not only increase
bioavailability of phytate phosphorus, but also to improve
zinc utilization as measured by plasma zinc concentrations,
plasma alkaline phosphatase activity, and growth

performance.

EXPERIMENTAL SERIES IV
INTERACTIONS OF PHYTASE, VITAMIN D, AND
CALCIUM ON PHYTATE PHOSPHORUS UTILIZATION

(Submitted to J. Anim. Sci.)

ABSTRACT

The objective of this study was to determine the effect
of dietary Ca and vitamin D on efficacy of microbial phytase
(A. niger) in improving phytate-P utilization. A 2 X 2 X 2
factorial arrangement of treatments was conducted with two
dietary levels of phytase (unit/g), 750 (suboptimal) and
1200 (optimal); of vitamin D (IU/kg), 660 (normal) and 6600
(high); and of Ca (%), .4 (low) and .8 (normal). Sixty-four
weanling pigs (4-wk-old, 8.04 i .50 kg BW) were allotted to
16 pens and fed the corn-soybean meal diets (no inorganic P
added) with variable levels of phytase, vitamin D, and Ca.
Individual pig weights and pen feed consumption were
measured at d 10, d 20, and d 30. Blood samples were taken
from each individual pig at the same time to assay plasma
inorganic P and Ca concentration and alkaline phosphatase
(AP) activity. The results indicated a strong (2 < .05)
adverse effect of normal dietary Ca on all the response
measures. The depressive effect of normal Ca on performance
was greater (P < .05) at normal vitamin D level or at
optimal phytase level than at the other level of these two

factors. The elevation in plasma AP activity by normal

115

116
dietary Ca was greater (2 < .05) at the suboptimal than at
the optimal phytase level. The decrease in plasma inorganic
P and increase in plasma Ca by normal dietary Ca were
overwhelming, but the magnitude of the changes still varied
with levels of vitamin D and phytase. Overall, the best
response of measures occurred when optimal phytase level was
combined with levels of low Ca and normal vitamin D. In
conclusion, normal level of Ca in the low-P, corn-soybean
meal diets greatly reduced the efficacy of supplemental
phytase. Raising vitamin D in the diets partially offset
this adverse effect, but did not produce further improvement
when Ca level was low.
Key Words: Pigs, Phytase, Vitamin D, Calcium, Phytate,

Phosphorus, Plasma Alkaline Phosphatase

Introduction

Supplementing swine diets with microbial phytase (Ay
pigsp) greatly improves phytate-P bioavailability and
decreases P excretion in the manure (Nasi, 1990; Simons et
al., 1990; Cromwell, 1991; Jongbloed et al., 1992; Leunissen
and Young, 1992). In a series of earlier studies, we
demonstrated that supplemental phytase at 1200 phytase units
(PU)/g of corn-soybean meal diets maximized phytate-P
utilization and essentially obviated the need for inorganic

P addition in weanling pigs (Lei et al., 1992b). However,

117
only a single dietary level of Ca and vitamin D, two key
nutrients which strongly interact with P (Littledike and
Goff, 1987) and affect phytate-P utilization (Wise, 1983),
was used in all these studies. High levels of Ca in the
diets of rats (Taylor and Coleman, 1979; Nahapetian and
Young, 1980) and poultry (Edwards and Veltman, 1983; Ballam
et al., 1985; Sheideler and Sell, 1987) consistently
decreased the availability of phytate-P. Likewise, vitamin D
deficiency was found to aggravate the disturbances of P and
Ca metabolism resulting from high dietary phytate-P
(Pointillart et al., 1985). In contrast, dietary supplements
of cholecalciferol improved phytate-P bioavailability to
pigs considerably (Fontaine et al., 1985). Recently,
Mohammed et al. (1991) reported that the adverse effects of
a high-phytate, low inorganic P diet on chicks were overcome
by the independent but synergistic effects of lowering Ca
and simultaneously raising cholecalciferol in the diets.
Therefore, we proposed that dietary Ca and vitamin D would
affect the efficacy of supplemental phytase by interacting
with phytate-P. This experiment was conducted to determine
the effect of dietary concentrations of Ca and vitamin D on

phytase improvement of phytate-P utilization by weanling

pigs.

Materials and Methods

Egperimental Design. This study was arranged as a 2 X 2
X 2 factorial in randomized complete blocks. Because of
repeated measurements over time, the entire design is a
split-block. The three treatment factors included dietary
phytase, Ca, and vitamin D. The two dietary levels of
phytase (PU/g) were set at 750 (suboptimal) and 1200
(optimal), of Ca (%) at .4 (low) and .8 (normal), and of
vitamin D (IU/kg) at 660 (normal) and 6600 (high).

Phytase and Diets. The microbial phytase (A. niger,
Alko Ltd., Rajamaki, Finland), the confirmation of the
actual activity of enzyme product, and the process of
phytase incorporation into diets were the same as described
previously (Lei et al., 1992a). A phytase unit (PU) is
defined as the amount of enzyme that liberates 1 nmol of
inorganic P from sodium phytate per minute at pH 5 and 37
°C. Calcium carbonate (Calcium Products, Inc., Swayzee, IN)
and vitamin E5 (Carl Akey, Lewisburg, OH) were used to
achieve the dietary levels of these two nutrients. The basal
diet (BD) was a fortified corn-soybean meal diet without
supplemental inorganic P. The composition and the analyzed
concentrations of P and Ca in the basal diet and the
experimental diets are presented in Table 4.1.

Animals. Sixty-four weanling crossbreds (Landrace-

Yorkshire-Hampshire, 4-wk-old) were grouped into heavy (8.91

118

119
i .61 kg BW) and light (7.17 r .36 kg BW) blocks based on
body weight. The 32 pigs within each block were allotted
into 8 pens of four pigs each. Housing, management, P-
depletion procedures, and the experimental periods were the
same as in previous studies (Lei et al., 1992a, b).

Sample Collection apg Measurements. Individual pig
weights and pen feed consumption were measured at d 10, d
20, and d 30. Blood samples were taken at the same frequency
from all pigs for assay of plasma inorganic P and Ca
concentrations and plasma alkaline phosphatase (AP) activity
(Lei et al., 1992a).

Statistics. Data were analyzed as a split-block model
with factorial treatments and time repeated measurements
(Gill, 1986). Pen was considered the experimental unit. As
recommended by Gill (1986), standard errors of differences
of means, instead of standard errors of single means, are
presented to overcome possible correlations induced by
repeated measurement. The Bonferroni p-test was used for
conditional comparisons of treatment means. Interactions
were considered to have modest importance if 2 < .30, but
primary comparisons were declared significant only for P <
.05, unless indicated otherwise.

Correlations between various response measures were

developed by the GLM procedure of SAS (1988).

120

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Results

Main Effects and Interactions of Treatments. The
Significances of main effects and their interactions of
dietary phytase, vitamin D, and Ca and of block are
summarized in Table 4.2. Dietary Ca significantly affected
all the response measures whereas vitamin D alone did not
affect any of these measures. Dietary phytase showed an
effect only on plasma Ca concentration (2 < .06). Marginal
interaction (2 < .12) of dietary phytase, vitamin D, and Ca
was observed on ADFI and plasma Ca concentrations. The same
type interaction but less significant (2 < .25) was also
shown on ADG and plasma AP activity. Dietary phytase and Ca
appeared to interact on gain/feed (P < .20), plasma AP
activity (P <. 25), and plasma Ca concentration (P < .29).
Similarly, dietary vitamin D seemed to interact with dietary
Ca on all the response measures except plasma AP activity.
In addition, body weight (block) had significant effect on
all the measures but gain/feed or plasma AP activity. The
standard errors of difference of two means for the main
effects and the interactions, and the calculated degrees of
freedom of error for each response criterion (Gill, 1986)

are presented in Table 4.3.

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124

Table 4.3. Standard errors of mean differences and degrees
of freedom for various measures

 

Main Two way Three way Error
Measure effect interaction interaction df
Daily gain, g 34.8 49.2 69.6 10.2
Feed intake, g/d 55.9 79.0 111.7 10.7
Gain/feed, g/kg 26.5 37.0 53.0 23.8
Plasma P, mg/dL .22 .31 .44 12.9
Plasma Ca, mg/dL .23 .32 .45 21.9
Plasma AP, U/dL .92 1.30 1.84 14.2

 

Daily Gainl Daily Feed Intake and GainzFee . The
effects of dietary phytase, vitamin D, and Ca on ADG, ADFI,

and gain/feed are presented in Table 4.4. Phytase at 750 or
1200 PU/g of the corn-soybean basal diets without added
inorganic P supported the pig growth at normal rate and
efficiency (NRC, 1988) when dietary Ca level was low (.4%).
In contrast, the effect of the same amount of phytase on
ADG, ADFI, and gain/feed was markedly reduced or limited at
the normal dietary Ca (.8%). Overall, pigs receiving the
normal level of dietary Ca had much lower ADG than those
receiving the low level of dietary Ca at d 10 (2 < .05), d
20 (g < .01), and d 30 (g < .01). This type of decrease in
ADG was much greater at the normal vitamin D level (P < .02)
than at the high vitamin D level, and was even intensified

at the optimal phytase level. At the combined optimal level

125

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126

of phytase and normal level of vitamin D, pigs receiving
the normal Ca level grew less than half the rate (2 < .05)
as those receiving the low Ca level. However, at otherwise
combined levels of phytase and vitamin D, the differences in
ADG between the two levels of Ca were not significant.
Similarly, ADFI was greatly suppressed at the normal dietary
Ca level, and the largest decline occurred at the combined
levels of optimal phytase and normal vitamin D. But, the
decrease was not significant until d 30. Poor feed
efficiency resulted from feeding the pigs with normal
dietary Ca just after d 10 (P < .01). The lowering effect of
normal dietary Ca on gain/feed was significant at the
optimal but not the suboptimal level of phytase. Likewise,
the same effect was significant at the normal but not the
high level of vitamin D. Unlike in the case of ADG or ADFI,
this decreasing effect did not vary with the three way
interaction. Compared to the suboptimal phytase level,
optimal phytase level tended to improve ADG, ADFI, and
gain/feed. Such improvement appeared to be more visible when
levels of vitamin D and Ca in the diets were either both low
or both high, though differences on the response measures
were never significant. In addition, the heavy pigs ingested
more feed (2 < .05) and thus grew faster (P_< .05) than the
light pigs.But, there was no difference (P_> .05) in

gain/feed between these two groups.

127

Plasma Inorganic P and Ca Concentrations and Alkaline
Phosphatase Activity. Effects of dietary phytase, vitamin D,
and Ca on plasma P and Ca concentrations and AP activity are
presented in Table 4.5. As shown previously (Lei et al.,
1992b), phytase at 750 or 1200 PU/g of the basal diets
resulted in a fairly normal plasma P status in pigs fed the
low level of dietary Ca. However, at the normal level of
dietary Ca, plasma P concentrations were decreased whereas
plasma Ca concentrations and AP activities were elevated.
Plasma P concentrations were lower (2_< .01) at the normal
level of Ca than at the low level Ca through the
entire study. This difference tended to be attenuated at the
optimal level of phytase and at the high level of vitamin D.
However, these interactions, either two way or three way,
were weak (2 > .35). As expected, plasma Ca concentrations
in pigs receiving the normal level of dietary Ca were higher
(P < .01) than those of pigs receiving the low level of
dietary Ca. Though these differences were overwhelming
across all levels of phytase and vitamin D, their magnitude
varied considerably because of a marginal three way
interaction of phytase, vitamin D, and Ca (2 < .12). When
optimal phytase level was combined with normal vitamin D
level, the differences in plasma Ca concentrations between

pigs fed the

128

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129
normal and low Ca levels were appreciably smaller than those
shown at other occasions. Besides, dietary phytase alone was
shown to reduce plasma Ca concentrations (2 < .05) at d 10
and d 30. High level of vitamin D in the diets tended to
enhance plasma Ca concentrations. Plasma AP activity was
elevated in the pigs receiving normal level of Ca compared
with that of pigs receiving the low level of Ca, but
differences were significant only at the suboptimal level of
phytase and at d 20 and d 30. Optimal phytase activity in
the diets appeared to minimize these differences to a
nonsignificant scale. Dietary levels of vitamin D had little
effect on plasma AP activity. In addition, heavy pigs had
higher (P < .05) plasma concentrations of P and Ca than the
light pigs. But plasma AP activity was not different between
these two groups of pigs.

Correlations among Various Response Measures. A strong
positive correlation (2 < .001) was found between ADG and
ADFI at d 10 (;_= .92), d 20 (r = .89), and d 30 (r = .90).
Moderate positive correlations (.5 < r < .8, P < .05) were
observed among ADG, ADFI, gain/feed, and plasma P
concentrations. Plasma P and Ca concentrations were
negatively correlated at d 10 (r = -.59, P < .02), d 20 (r =
-.71, 2 < .002), and d 30 (r = -.71, P < .002). Likewise,
plasma P concentrations and AP activity were also inversely
correlated at d 20 (g = -.52, P < .04) and d 30 (g = -.53, E

< .03). Plasma Ca concentrations and AP activity were

130

positively correlated at d 20 (r = .60 , g < .01).

Discussion

Dietary P, phytate, Ca, and vitamin D are closely
related in metabolism in the animal body (Wise, 1983;
Pointillart et al., 1984, 1985, 1987). But recent studies on
effect of microbial phytase on utilization of phytate-P by
pigs during starting (Lei et al., 1992 a, b; Leuniessen and
Young, 1992) and growing-finishing phases (Nasi, 1990;
Simons et al., 1990; Cromwell, 1991; Jongbloed et al., 1992)
were almost exclusively conducted with a single level of Ca
or vitamin D. In most cases, dietary Ca level was relatively
low. In the present study, the efficacy of phytase on
improving phytate-P availability was greatly reduced at the
normal level of dietary Ca compared to that at the low level
of dietary Ca. When all pigs were fed the corn-soybean meal
diets without added inorganic P, supplementing phytase at
either 750 or 1200 PU/g feed supported only pigs receiving
low Ca to perform at a normal rate of gain (NRC, 1988) and
to maintain normal plasma P status, as shown previously (Lei
et al., 1992b). In contrast, the same amount of phytase
activity in the diets with normal Ca concentration (.8%)
failed to produce the same improvements, as indicated by
appreciably lower ADG, ADFI, and gain/feed and unfavorable

changes in plasma inorganic P and Ca concentrations and AP

131
activity. Calcium, the major divalent cation in the diets
for many species, can progressively precipitate all the
phytate by forming extremely insoluble Ca-phytate complex in
the intestine (Wise, 1983; Nelson and Kirky, 1987).
Consequently, phytate-P, as well as Ca itself, is largely
unavailable to digestion (Wise, 1983). It has been
conclusively demonstrated that high levels of Ca in the
diets of rats (Taylor and Coleman, 1979; Nahapetian and
Young, 1980) and of poultry (Edwards and Veltman, 1983;
Ballam et al., 1985; Sheideler and Sell, 1987) decreased the
availability of phytate-P considerably. Lowering dietary Ca
from 1% to .5% enhanced phytate-P digestibility by 15% in
chicks (Mohammed et al., 1991). However, the data on the
effects of dietary Ca on phytate-P utilization by pigs were
scarce (Pointillart et al., 1989). Jongbloed (1987) reported
that absorption of P in diets without supplemental inorganic
P was inversely related to dietary Ca up to .65%. Meanwhile,
retention of dietary P was improved by the increase in
dietary Ca. Pointillart et al. (1989) found that elevating
dietary Ca from .9% to 1.4% in the diets containing .5% P
(all from plant) intensified the P deficiency secondary to
high phytate feeding. But they failed to observe a
detrimental effect of high dietary Ca on phytate-P
absorption or retention and suggested that the absence of
decreased P absorption might result from the appropriate

dietary vitamin D level (1,000 IU/kg).

132

The effect of dietary Ca on performance and plasma P
status varied considerably with dietary vitamin D level,
though vitamin D alone had no significant effects on any of
the response measures. The high level of vitamin D in the
diets appeared to reduce the adverse effects of normal
dietary Ca on ADG, ADFI, gain/feed, and plasma inorganic P
concentrations. This type of interaction between vitamin D
and Ca was in agreement with that observed by Mohammed et
al. (1991). They found that introduction of a high level of
cholecalciferol (10 fold higher than normal) to the chick
diets led to a marked increase in circulating levels of
1,25-(0H)§§. As a result, availability of Ca and phytate
were significantly increased, and the hypophosphatemia
associated with the low P intake was alleviated. Likewise,
absorption or retention of P in a diet for pigs with .6% P
(80% Phytate—P) and .6% Ca was virtually doubled when the
diet was supplemented with cholecalciferol at 1000 IU/kg
(Fontaine et al., 1985). However, the formation of
unavailable Ca-phytate, suggesting by a simultaneous
increase in fecal Ca and P excretion (; > .92, 2 < .05)with
time, took place in vitamin D-depleted pigs (Pointillart et
al., 1985). As vitamin D supplementation was shown to have
no effect on phytase or AP activity in intestinal mucosa of
pigs (Fontaine et al., 1985), it may oppose the depressive
effect of Ca in phytate-P availability by activating Ca

absorption and thus preventing, or at least reducing, Ca-

133
phytate formation (Pointillart et al., 1989). On the other
hand, increasing dietary Ca from .5% to 1.0% did not
influence 1,25-(OH)5§ levels in plasma of chicks fed low-P
diets (Mohammed et al., 1991).

Phytase level also influenced the action of dietary Ca.
Optimal level of phytase in the diets appeared to magnify
the differences in perforamce measures between the two
levels of dietary Ca. But optimal level of phytase tended to
minimize the same type of differences in plasma AP activity,
and to a lesser extent in plasma inorganic P or Ca
concentrations. Based on the relationship between dietary
phytase activity and plasma measures (Lei et al., 1992a, b)
and the correlations among these measures shown in this
study, we would expect higher levels of phytase to free more
P from phytate and then to reduce the resultant elevation in
plasma AP activity. Superficially, it may look like to be
contradictory that optimal phytase alleviated the adverse
effect of dietary Ca on plasma P status, but aggravated the
detrimental effect of dietary Ca on performance. However, if
we assume that the effect of normal dietary Ca on phytate
was certain, the differences in performance between the two
levels of Ca then would mainly depend on the effect of
phytase. Just as previously shown (Lei et al., 1992b),
phytase at 1200 PU/g feed resulted in greater improvement in
performance at the low dietary Ca than phytase at 750 PU/g

feed. On the other hand, the differences in performance

134
measures between the two levels of phytase at normal dietary
Ca were relatively small. Thus, the differences between the
two levels of dietary Ca became significant at the optimal
level but not at the suboptimal level of phytase. As phytase
was provided in the diets, no severe P deficiency developed.
Only plasma P concentrations were significantly related to
performance measures with modest coefficient. Besides, the
differences in most measures between the two levels of
phytase were relatively smaller than those observed
previously (Lei et al., 1992b). Some interactions between
phytase and Ca or vitamin D may have somewhat confounded the
effect of phytase.

Among the eight combinations of the three dietary
treatments, optimal phytase combined with normal vitamin D
and low Ca seemed to produce the best overall response.
Simultaneous lowering of Ca and elevation of vitamin D in
the diets did not cause additive benefits compared to singly
reducing dietary Ca. This was different from what was
observed by Mohammed et al. (1991). They found that
simultaneous lowering of dietary Ca and elevation of vitamin
D in the low-P diets for chicks additively improved phytate-
P utilization. However, when microbial phytase was
incorporated into the diets in this study, dietary Ca rather

than vitamin D became more crucial.

Implications

Supplementing corn-soybean meal diets with microbial
phytase greatly improves phytate phosphorus utilization and
thus essentially obviates the need for inorganic phosphorus
supplementation. However, this can only be achieved at
moderately low levels of dietary calcium. Supplying calcium
at a dietary level normally recommended or used results in
an appreciable decrease in phytase efficacy. Introducing
high levels of vitamin D in the diets may partially offset
this adverse effect of Ca, but does not further improve
phytate phosphorus utilization in low Ca diets with

microbial phytase.

135

 

GENERAL DISCUSS ION

GENERAL DISCUSSIONS

Progress in this Research

The results of the four consecutive studies on
supplemental A. niger phytase in diets for weanling pigs
lead to the followings conclusions:

1. Supplementing the enzyme up to 750 PU/g of a low-P,
corn-soybean meal basal diet (BD) increased P retention by
50% and decreased fecal P excretion by 42%. Raising the
enzyme activity to 1,200 or 1,350 PU/g of the BD resulted in
relatively greater improvements in these two measures.

2. Plasma inorganic P concentrations, ADG, and ADFI
increased linearly with dietary phytase activity from 0 to
750 PU/g of the BD.

3. Responses of ADG, ADFI, and plasma alkaline
phosphatase activity maximized at approximately 1,200 PU/g
of the BD. However, plasma inorganic P and Ca concentrations
kept linearly increasing and decreasing, respectively, up to
1,350 PU/g of the BD.

4. One thousand units of phytase activity supported the
retention of 1.1 mg P from the BD and were equivalent in

effect to .91 mg P from mono-dibasic calcium phosphate. Pigs

136

137
receiving the BD with phytase at 1,200 PU/g retained
virtually the same amount of P as those receiving the
inorganic P supplemented control diets.

5. Plasma concentrations of inorganic P and Ca and
daily urinary excretions of P and Ca were moderately
correlated. High concentrations of plasma inorganic P in
pigs supplemented with inorganic P in their diets led to
increased daily urinary excretions of P.

6. Supplementing this enzyme at 1,350 PU/g of a corn-
soybean meal diet without added inorganic forms of P and Zn
appeared to release sufficient Zn and phytate-P from the
diet to maintain normal status of plasma inorganic P and Zn
and normal rate and efficiency of gain.

7. Utilization of dietary Ca was also improved by
supplemental phytase. However, different levels of phytase
did not influence plasma concentrations of Mg, Cu, Fe, and
Zn when these elements were provided at the recommended
levels in the diets.

8. All improvements in the various measures by adding
phytase to the diets as described above were achieved at
reduced dietary Ca levels. Normal Ca in the diets greatly
suppressed the efficacy of phytase. Raising vitamin D level
in the diets partially offset this adverse effect. It
appears that the optimal Ca needed for maximum efficacy of
supplemental dietary phytase is different from the metabolic

Ca requirement.

138

Part of the above conclusions was also supported by the
results of other researchers (Nasi, 1990; Simons et al.,
1990; Cromwell, 1991; Jongbloed et al., 1992; Leunissen and
Young, 1992). However, their approaches were quite different
from that used in this research. Researchers in Europe
(Nasi, 1990; Simons et al., 1990; Jongbloed et al., 1992)
studied the effects of A. niger phytase on phytate-P and
total P digestion and(or) retention. But, they did not have
an inorganic P supplemented diet as a positive control. In
addition, only a limited number of pigs were tested, and
effect of phytase on performance, plasma inorganic P status
or bone traits was not determined or reported. Researchers
at the University of Kentucky (Cromwell, 1991) compared the
effects of two levels of phytase (500 and 1,000 PU/g) on
performance and bone strength. Inorganic P supplemented
diets were used as a positive control. However, plasma
inorganic P status and P balance were not measured.
Leunissen and Young (1992) of the University of Guelph
compared the effect of 500 and 1,000 PU/g feed with that of
regular inorganic P supplementation on all the four major
response measures taken by the above two groups. However,
their basal diet was not simply composed of corn and soybean
meal and small portion of inorganic P was supplemented.
Again, only a limited number of pigs were tested at a fairly
short period (3 wk). In comparison, four major experiments

were conducted consecutively with a total of 258 pigs in

139
this research. A series of 7 graded levels of phytase
activity was compared. Both negative control (the BD) and
positive control (inorganic P supplements) were used.
Measurements included performance, plasma mineral
concentrations and alkaline phosphatase activity, and
mineral balance. Systematic data were generated from fairly
large samples and thereby were very consistent. Furthermore,
the simultaneous improvement in Zn and phytate-P utilization
by phytase were demonstrated. Interactions of dietary

phytase, vitamin D, and Ca were also determined.

Applications of this Research

The followings implications may be inferred from the
above conclusions:

1. Supplementing A. niger phytase at 1,200 PU/g of
corn-soybean meal diets essentially obviates the need for
inorganic P supplementation. Thus, diet cost would be
decreased and large amounts of inorganic P resources would
be saved.

2. Replacing inorganic P by phytase in the diets would
eliminate, at least, alleviate the P pollution originating
from the excess P in the swine manure in areas of intensive
animal production.

3. Supplementing phytase may reduce the Zn requirement

of pigs. This enzyme may also be used to improve

140
bioavailability of Zn in infant soy formula and to treat
other foods of plant origin consumed by humans.

4. Dietary Ca level should be reduced to permit full
efficacy of supplemental phytase.

In addition, Cromwell (1991) suggested that
supplementing phytase at 1,000 PU/g of corn-soybean meal
diets could replace all the inorganic P supplementation in
finishing pigs. Jongbloed and Kemme (1990) demonstrated that
proper pelleting feeds with phytase did not reduce the
enzyme activity. If all these results were applied in swine
production, major impact in nutrition and environment could

be expected.

Limitations of this Research

The major concern associated with the interpretation
and application of this research results was a possible
carry-over effect. Low-P feeding of weanling pigs may be
detrimental to their later development and(or) performance,
particularly to breeding herds. The lack of testing phytase
on bone traits amplify this concern. In all, no economical
source of phytase is available. Supplementing phytase at the
optimal level shown in this research would be more expensive
than the routine addition of different sources of inorganic
P. Therefore, future use of phytase in swine production

mainly depends on the success of continuous seeking high

 

 

141
producers of phytase and(or) improving the enzyme production
by biotechnology. Optimizing dietary conditions may maximize
phytase efficacy and then reduce the required level of the

enzyme and facilitate the economy of phytase usage.

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